Antimicrobial methods and materials

ABSTRACT

The present invention provides methods of identifying agents that bind gene products critical for the survival of microbes, preferably Staphylococcus microbes, including agents that interfere with the expression of such gene products and agents that interfere with the function of such gene products.

BACKGROUND

[0001] The staphylococci, of which Staphylococcus aureus is the mostimportant human pathogen, are hardy, gram-positive bacteria thatcolonize the skin of most humans. Staphylococcal strains that producecoagulase are designated S. aureus; other clinically importantcoagulase-negative staphylococci are S. epidermidis and S.saprophyticus. When the skin or mucous membrane barriers are disrupted,staphylococci can cause localized and superficial infections that arecommonly harmless and self-limiting. However, when staphylococci invadethe lymphatics and the blood, potentially serious complications mayresult, such as bacteremia, septic shock, and serious metastaticinfections, including endocarditis, arthritis, osteomyelitis, pneumoniaand abscesses in virtually any organ. Certain strains of S. aureusproduce toxins that cause skin rashes, food poisoning, or multisystemdysfunction (as in toxic shock syndrome). S. aureus and S. epidermidistogether have become the most common cause of nosocomial non-urinarytract infection in U.S. hospitals. They are the most frequently isolatedpathogens in both primary and secondary bacteremias and in cutaneous andsurgical wound infections. See generally Harrison's Principles ofInternal Medicine, 13th ed., Isselbacher et al., eds., McGraw-Hill, NewYork (1994), particularly pages 611-617.

[0002] Transient colonization of the nose by S. aureus is seen in 70 to90 percent of people, of which 20 to 30 percent carry the bacteria forrelatively prolonged periods of time. Independent colonization of theperineal area occurs in 5 to 20 percent of people. Higher carriage ratesof S. aureus have been documented in persons with atopic dermatitis,hospital employees, hospitalized patients, patients whose care requiresfrequent puncture of the skin, and intravenous drug abusers.

[0003] Infection by staphylococci usually results from a combination ofbacterial virulence factors and a diminution in host defenses. Importantmicrobial factors include the ability of the staphylococcus to surviveunder harsh conditions, its cell wall constituents, the production ofenzymes and toxins that promote tissue invasion, its capacity to persistintracellularly in certain phagocytes, and its potential to acquireresistance to antimicrobial agents. Important host factors include anintact mucocutaneous barrier, an adequate number of functionalneutrophils, and removal of foreign bodies or dead tissue.

[0004] Cell wall components of S. aureus include a large peptidoglycancomplex that confers rigidity on the organism and enables it to surviveunder unfavorable osmotic conditions, a unique teichoic acid linked topeptidoglycan, and protein A, which is found both attached topeptidoglycan over the outermost parts of the cell and released insoluble form. Proteins designated femA and femB are involved in theformation of cell wall peptidoglycan pentaglycine cross-bridges and arefactors in methicillin resistance (Berger-Bachi et al., Mol. Gen.Genet., 219, 263-269 (1989)). S. aureus also has specific receptors forlaminin and fibronectin that may mediate the organism's spread throughthe bloodstream to other tissues. Both peptidoglycan and teichoic acidare capable of activating the complement cascade via the alternativepathway. S. aureus also appears to activate tissue factor in thecoagulation pathway.

[0005] Certain enzymes produced by S. aureus may play a role invirulence. Catalase degrades hydrogen peroxide and may protect theorganism (luring phagocytosis. Coagulase is present in both soluble andcell-bound forms and causes plasma to clot by formation of thrombin-likematerial. The high correlation between coagulase production andvirulence suggests that this substance is important in the pathogenesisof staphylococcal infections, but its precise role as a determinant ofpathogenicity has not been determined. Many strains also producehyaluronidase, an enzyme that degrades hyaluronic acid in the connectivetissue matrix and that may promote spreading of infection. Atrypsin-like protease from some strains enhances influenza virusinfection by proteolytic cleavage of the viral precursor hemagglutinininto its active fragments and may contribute to the morbidity of suchco-infections. S. aureus produces numerous extracellular exotoxins thathave been implicated in disease processes. The exfoliatin toxins A andB, the staphylococcal enterotoxins, and the toxic shock syndrome toxin,TSST-1, belong to the growing family of microbial superantigens thatactivate T cells and monocytes/macrophages, resulting in the productionof cytokines that mediate local or systemic effects depending on theamount of toxin formed, the immune status of the host, and the access ofthe toxin to the circulation. The exfoliatin toxins mediate thedermatologic manifestations of the staphylococcal scalded-skin syndromeand bullous impetigo. These toxins cause intraepidermal cleavage of theskin at the stratum granulosum, leading to bullae formation anddenudation. Seven distinct enterotoxins (A, B, C1, C2, C3, D, and E)have been implicated in food poisoning due to S. aureus. These toxinsenhance intestinal peristalsis and appear to induce vomiting by a directeffect on the central nervous system. Toxic shock syndrome (TSS) is mostcommonly mediated by TSST-1, which is present in 5 to 25 percent ofclinical isolates of S. aureus. TSS is also mediated less frequently byenterotoxin B and, rarely, enterotoxin C1.

[0006]S. aureus produces other toxins whose role in virulence isincompletely understood. Four different red blood cell hemolysins, whichare designated alpha, beta, gamma, and delta toxins, have beenidentified. Alpha toxin also causes necrosis of the skin when injectedsubcutaneously into animals, while delta toxin also inhibits waterabsorption in the intestines and may play a role in the acute waterydiarrhea seen in some cases of staphylococcal infection. Leukocidinlyses granulocyte and macrophage membranes by producing membrane porespermeable to cations.

[0007] The agr, xpr, sae and sar coding sequences have been identifiedas being involved in the regulation of staphylococcal exotoxins. SeeU.S. Pat. No. 5,587,228 and International Patent Publication Nos. WO96/10579 and WO 97/11690. Of interest is the report in WO 97/11690 ofscreening for inhibitors of these regulatory systems.

[0008] Staphylococci can invade the skin or mucosa through plugged hairfollicles and sebaceous glands or areas traumatized by bums, wounds,abrasions, insect bites, or dermatitis. Staphylococci often colonizeprosthetic devices and intravenous catheters; S. aureus infection of thevascular access site is a major cause of morbidity and death amongpatients on hemodialysis. Colonization and invasion of the lungs mayoccur with endotracheal intubation, or when the lungs' clearancemechanisms are depressed, e.g., after viral infections, afteraspiration, or in patients with cystic fibrosis. Mucosal damage to thegastrointestinal tract following cytotoxic chemotherapy or radiotherapypredisposes to invasion from that site.

[0009] Once the skin or mucosa have been breached, local bacterialmultiplication is accompanied by inflammation, neutrophil accumulation,tissue necrosis, thrombosis and fibrin deposition at the site ofinfection. Later, fibroblasts create a relatively avascular wall aboutthe area. When host mechanisms fail to contain the cutaneous orsubmucosal infection, staphylococci may enter the lymphatics and thebloodstream. Common sites of metastatic spread include the lungs,kidneys, cardiac valves, myocardium, liver, spleen, bones and brain.

[0010] Bacteremia due to S. aureus may arise from any local infection,at either extravascular (cutaneous infections, bums, cellulitis,osteomyelitis, arthritis) or intravascular foci (intravenous catheters,dialysis access sites, intravenous drug abuse). Commonly, the diseaseprogresses more slowly, with hectic fever and metastatic abscessformation. Rarely, patients with bacteremia die within 12 to 24 hourswith high fever, tachycardia, cyanosis, and vascular collapse.Disseminated intravascular coagulation may produce a disease mimickingmeningococcemia.

[0011] A major complication of S. aureus bacteremia is endocarditis. S.aureus is the second most common cause of endocarditis and the mostcommon cause among drug addicts. The disease is typically acute, withhigh fever, progressive anemia, and frequent embolic and extracardiacseptic complications. Valve ring and myocardial abscesses are common.The mortality rate is 20 to 30 percent.

[0012] Staphylococcal scalded-skin syndrome (SSSS) is a generalizedexfoliative dermatitis that is a complication of infection by exfoliatintoxin-producing strains of S. aureus. The disease typically occurs innewborns (Ritter's disease) and in children under the age of five. Ascarlatiniform rash begins in the perioral area, becomes generalizedover the trunk and extremities, and finally desquamates. The disease mayconsist of rash alone (staphylococcal scarlet fever), or large, flaccidbullae develop that may be localized (more common in adults) orgeneralized. The bullae burst, resulting in red, denuded skin resemblinga burn. Most adults with SSSS are immunosuppressed or have renalinsufficiency. Blood cultures are frequently positive, and mortality issignificant.

[0013] Toxic shock syndrome (TSS) is a multisystem disease mediated bytoxins (generally TSST-1, and less frequently enterotoxins B and C1)produced by certain strains of S. aureus. It was first described inchildren, but in 1980 became epidemic among young women, with onsetduring menstruation. The diagnosis of TSS is based on clinical criteriathat include high fever, a diffuse rash that desquamates on the palmsand soles over the subsequent one or two weeks, hypotension that may beorthostatic, and evidence of involvement in three or more organ systems.Such involvement commonly includes gastrointestinal dysfunction(vomiting or diarrhea), renal or hepatic insufficiency, mucous membranehyperemia, thrombocytopenia, myalgias with elevated creatinephosphokinase (CK) levels, and disorientation with a normalcerebrospinal fluid examination. The mortality rate of TSS is threepercent.

[0014]S. aureus causes approximately three percent of community-acquiredbacterial pneumonias. This disease occurs sporadically except duringinfluenza outbreaks, when staphylococcal pneumonia is relatively morecommon, although still less frequent than pneumococcal pneumonia.Primary staphylococcal pneumonia in infants and children frequentlypresents with high fever and cough. Multiple thin-walled abscesses areseen on the chest X-ray, and empyema formation is common. In olderchildren and healthy adults, staphylococal pneumonia is generallypreceded by an influenza-like respiratory infection. Onset ofstaphylococcal involvement is abrupt, with chills, high fever,progressive dyspnea, cyanosis, cough, pleural pain, and sometimes bloodysputum. Staphylococcal pneumonia is seen more frequently in patientswith cystic fibrosis, in intubated patients in intensive care units andin debilitated patients who are prone to aspiration.

[0015]S. aureus is responsible for the majority of cases of acuteosteomyelitis. Although the disease is most common in people under theage of 20, it is becoming increasingly prevalent in adults over 50,particularly with involvement of the spine. A primary portal of entry isfrequently not identified, although many patients give a history ofpreceding trauma to the involved area. Once established, infectionspreads through the bone to the periosteum or along the marrow cavity.Rarely, the joint capsule is penetrated, producing pyogenic arthritis.Osteomyelitis in children may present as an acute process beginningabruptly with chills, high fever, nausea, vomiting, and progressive painat the site of bony involvement.

[0016]S. aureus causes 1 to 9 percent of cases of bacterial meningitisand 10 to 15 percent of brain abscesses. Most commonly, the bacteria arespread from a focus outside the central nervous system, typically frominfective endocarditis, by extension from a paraspinal or parameningealabscess, or by nosocomial infection following neurosurgical procedures.Over 50 percent of epidural abscesses are due to S. aureus; up to halfof these cases may be associated with vertebral osteomyelitis. Patientspresent with either acute or chronic back pain, usually with low-gradefever and malaise. The onset of radicular pain is an ominous sign thatthe disease may progress to neurologic dysfunction and ultimateparalysis.

[0017] Antimicrobial resistance by staphylococci favors theirpersistence in the hospital environment. Over 90 percent of bothhospital and community strains of S. aureus causing infection areresistant to penicillin. This resistance is due to the production ofβ-lactamase enzymes; the nucleotides encoding these enzymes are usuallycarried by plasmids. Infections due to organisms with such acquiredresistance can sometimes be treated with penicillinase-resistantβ-lactam antimicrobial agents. However, the true penicillinase-resistantS. aureus organisms, called methicillin-resistant S. aureus (MILSA), areresistant to all the β-lactam antimicrobial agents as well as thecephalosporins. MRSA resistance is chromosomally mediated and involvesproduction of an altered penicillin-binding protein (PBP 2a or PBP 2′)with a low binding affinity for β-lactams. MRSA frequently also haveacquired plasmids mediating resistance to erythromycin, tetracycline,chloramphenicol, clindamycin, and aminoglycosides. MRSA have becomeincreasingly common worldwide, particularly in tertiary-care referralhospitals. In the United States, approximately 5 percent of hospitalisolates of S. aureus are methicillin-resistant.

[0018] Thus, there continues to exist a need for new agents useful fortreating bacterial infections, particularly those caused byantibiotic-resistant bacteria, and for methods of identifying such newagents. Such methods ideally would identify agents that are unrelated toexisting antimicrobials and that target different aspects ofstaphylococcal invasion of and replication in the host, compared toexisting antimicrobials.

SUMMARY OF THE INVENTION

[0019] The present invention provides a method for identifying an agentthat binds a polypeptide. The method includes contacting a polypeptideand an agent to form a mixture, and determining whether the agent bindsthe polypeptide. In some aspects, the polypeptide may be encoded by acoding sequence having a nucleotide sequence of SEQ ID NO: 7, 21, 23,25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137, or 141. Inanother aspect of the invention, the polypeptide is encoded by anessential coding sequence having a nucleotide sequence with at leastabout 57 percent structural similarity to a nucleotide sequence of SEQID NO: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133,or 137. In another aspect, the polypeptide is encoded by a criticalcoding sequence having a nucleotide sequence with at least about 57percent structural similarity to the nucleotide sequence SEQ ID NO: 141.

[0020] Determining whether the agent binds the polypeptide may beaccomplished by conducting an enzyme assay, a binding assay, or a ligandbinding assay. The method may further include determining whether theagent decreases the growth rate of a microbe. This includes contacting amicrobe with the agent, incubating the microbe and the agent underconditions suitable for growth of the microbe that is not contacted withthe agent, and determining the growth rate of the microbe contacted withthe agent. A decrease in growth rate compared to the microbe that is notcontacted with the agent indicates the agent decreases the growth rateof the microbe. The microbe may be in vitro or in vivo, and the microbemay be a Staphylococcus aureus. The present invention also provides anagent identified by the method.

[0021] The present invention also provides a method for identifying anagent that decreases the growth rate of a microbe. The method includescontacting a microbe with an agent, incubating the microbe and the agentunder conditions suitable for growth of the microbe that is notcontacted with the agent, and determining the growth rate of the microbecontacted with the agent. A decrease in growth rate compared to themicrobe that is not contacted with the agent indicates the agentdecreases the growth rate of the microbe. In some aspects, thepolypeptide may be encoded by a coding sequence having a nucleotidesequence of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117,121, 125, 129, 133, 137, or 141. In another aspect of the invention, thepolypeptide is encoded by an essential coding sequence having anucleotide sequence with at least about 57 percent structural similarityto a nucleotide sequence of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 33,109, 113, 117, 121, 125, 129, 133, or 137. In another aspect, thepolypeptide is encoded by a critical coding sequence having a nucleotidesequence with at least about 57 percent structural similarity to thenucleotide sequence SEQ ID NO: 141. The microbe may be in vitro or invivo, and the microbe may be a Staphylococcus aureus. The presentinvention also provides an agent identified by the method.

[0022] Also provided by the present invention is a method for decreasingthe growth rate of a microbe. The method includes contacting a microbewith an agent that binds to a polypeptide. In some aspects, thepolypeptide may be encoded by a coding sequence having a nucleotidesequence of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117,121, 125, 129, 133, 137, or 141. In another aspect of the invention, thepolypeptide is encoded by an essential coding sequence having anucleotide sequence with at least about 57 percent structural similarityto a nucleotide sequence of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 33,109, 113, 117, 121, 125, 129, 133, or 137. In another aspect, thepolypeptide is encoded by a critical coding sequence having a nucleotidesequence with at least about 57 percent structural similarity to thenucleotide sequence SEQ ID NO: 141. The microbe may be in vitro or invivo, and the microbe may be a Staphylococcus aureus.

[0023] The present invention provides a method for making an S. aureuswith reduced virulence. The method includes altering a coding sequencein an S. aureus to include a mutation, and determining if the S. arueushaving the mutation has reduced virulence compared to an S. arueus thatdoes not have the mutation. The coding sequence that is altered toinclude a mutation may include a nucleotide sequence of SEQ ID NO: 7,21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137, or141. In another aspect of the invention, the coding sequence that isaltered to include a mutation is an essential coding sequence that mayinclude a nucleotide sequence having at least about 57 percentstructural similarity to a nucleotide sequence of SEQ ID NO: 7, 21, 23,25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133,or 137. In yetanother aspect, the coding sequence that is altered to include amutation is a critical coding sequence that may include a nucleotidesequence having at least about 57 percent structural similarity to anucleotide sequence of SEQ ID NO: 141. The invention also provides theS. aureus having reduced virulence, and a vaccine composition thatincludes the S. aureus having reduced virulence.

[0024] The present invention further provides isolated polynucleotides.A polynucleotide may include a nucleotide sequence of SEQ ID NO: 7, 21,23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137, or 141.In another aspect, a polynucleotide may include a nucleotide sequencehaving at least about 57 percent structural similarity with a nucleotidesequence of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 109, 113, 117, 121,125, 129, 133, or 137, wherein the isolated polynucleotide includes anessential coding sequence. In yet another aspect, a polynucleotide mayinclude a nucleotide sequence having at least about 57 percentstructural similarity with a nucleotide sequence of SEQ ID NO: 141,wherein the isolated polynucleotide includes a critical coding sequence.A polynucleotide may also consist essentially of the above describednucleotide sequences, and the polynucleotide may optionally furtherinclude from zero to up to about 5,000 nucleotides upstream and/ordownstream of the nucleotide sequence.

[0025] Also provided by the present invention are isolated polypeptidesthat include an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 110, 114, 118, 122, 126,130, 134, 138, or 142.

Definitions

[0026] As used herein, the term “agent” refers to chemical compounds,including, for instance, an organic compound, an inorganic compound, ametal, a polypeptide, a non-ribosomal polypeptide, a polyketide, or apeptidomimetic compound that binds to a particular polypeptide.

[0027] As used herein, the term “polypeptide” refers to a polymer ofamino acids and does not refer to a specific length of a polymer ofamino acids. Thus, for example, the terms peptide, oligopeptide,protein, and enzyme are included within the definition of polypeptide.This term also includes post-expression modifications of thepolypeptide, for example, glycosylations, acetylations, phosphorylationsand the like.

[0028] The term “binds to a polypeptide” refers to a condition ofproximity between an agent and a polypeptide. The association may benon-covalent, wherein the juxtaposition is energetically favored byhydrogen bonding, van der Waals forces, or electrostatic interactions,or it may be covalent.

[0029] As used herein, growth of a microbe “in vitro” refers to growth,for instance, in a test tube or on an agar plate. Growth of a microbe“in vivo” refers to growth, for instance, in a cultured cell or in ananimal.

[0030] As used herein, the term “microbe” and “bacteria” are usedinterchangeably and include single celled prokaryotic and lowereukaryotic (e.g., fungi) organisms, preferably prokaryotic organisms.

BRIEF DESCRIPTION OF THE FIGURES

[0031]FIGS. 1a-z. The nucleotide sequence of the coding sequences of 26S. aureus coding sequences (SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137,and 141), the predicted sequence of the peptide (SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 110, 114, 118, 122,126, 130, 134, 138, or 142, respectively) encoded by each codingsequence, and primer pairs used for preparing fragments for insertioninto a temperature sensitive plasmid (SEQ ID NO: 35-68, 111-112,115-116, 119-120, 123-124, 127-128, 131-132, 135-136, 139-140, and143-144). The two underlined sequences in each coding sequencecorrespond to the primers listed below the coding sequence.

[0032]FIGS. 2a-i. The nucleotide sequence of each of 9 S. aureus codingsequences to be cloned for expression in E. coli (SEQ ID NO: 69, 71, 73,75, 77, 79, 81, 83, and 85), the predicted sequence of the peptide (SEQID NO: 70, 72, 74, 76, 78, 80, 82, 84, and 86, respectively) encoded byeach coding sequence after insertion into the appropriate expressionplasmid, and the sequence of the primer pair (SEQ ID NO: 91-108) used toclone the S. aureus coding sequences by amplification. The top primerand bottom primer of each primer pair is the forward primer and thereverse primer, respectively. The underlined ATGG in SEQ ID NO: 69, 73,75, 77, and 79 shows the location of a portion of the NcoI restrictionsite added to the coding sequence by the forward primer for cloning intothe expression vector pQE-60. The underlined AGATCT in SEQ ID NOS: 69,71, 73, 75, 79, 81, 83, and 85 shows the location of the BglIIrestriction site added to the coding sequence by the reverse primer. Theunderlined GGATCT in SEQ ID NO: 77 shows the location of the ligation ofthe digested BamHI restriction site of the amplified fragment with thedigested BglII restriction site of the vector.

[0033]FIG. 3. Nucleotide sequence (SEQ ID NO: 87) and the predictedamino acid sequence (SEQ ID NO: 88) of the S. aureus uridylate kinase.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

[0034] The sequence of the S. aureus genome has been determined andincludes about 3,500 coding sequences (see, for instance, Kunsch et al.,EP 0 786 519 A2). As used herein, the terms “coding sequence,” “codingregion,” and “open reading frame” are used interchangeably herein andrefer to a nucleotide sequence that encodes a polypeptide and, whenplaced under the control of appropriate regulatory sequences, expressesthe encoded polypeptide. The boundaries of a coding region are generallydetermined by a translation start codon at its 5′ end and a translationstop codon at its 3′ end. A regulatory sequence is a nucleotide sequencethat regulates expression of a coding region to which it is operablylinked. Nonlimiting examples of regulatory sequences include promoters,transcription initiation sites, translation start sites, translationstop sites, and terminators. “Operably linked” refers to a juxtapositionwherein the components so described are in a relationship permittingthem to function in their intended manner. A regulatory sequence is“operably linked” to a coding region when it is joined in such a waythat expression of the coding region is achieved under conditionscompatible with the regulatory sequence.

[0035] At this time, it is not possible to predict the function of someof the polypeptides that the approximately 3,500 coding sequences of theS. aureus genome are predicted to encode. This subset of codingsequences are referred to herein as “unknown coding sequences.” Amongthe large number of unknown coding sequences in the S. aureus genome,those that are required for cell growth are potential novel targets forantimicrobial therapy. The function of other coding sequences of the S.aureus genome can be hypothesized by comparing an S. aureus codingsequence with a second coding sequence from another organism, where thesecond coding sequence has a known function. This subset of codingsequences are referred to herein as “known coding sequences.” However,even though the function of these coding sequences can be hypothesized,for many it is unknown if they are required for bacterial growth. Thoseknown coding sequences that are required for bacterial growth arepotential novel targets for antimicrobial therapy.

[0036] As used herein, a “critical coding sequence” encodes apolypeptide that is required for a bacterial cell, preferably S.epidermidis, S. saprophyticus, or S. aureus, more preferably, an S.aureus cell, to grow at a normal growth rate in vitro or in vivo,preferably in vitro. Such polypeptides are referred to herein as“critical polypeptides.” A coding sequence is a critical coding sequencewhen mutagenesis of the coding sequence in a bacterial cell decreasesthe growth rate of the bacterial cell to, in increasing levels ofpreference, less than about 50%, less than about 60%, less than about80%, most preferably, less than about 90% of the growth rate of thebacterial cell that does not contain the mutated coding sequence.Methods of measuring the growth rate of microbes are well known androutine in the art. A critical coding sequence may encode a polypeptidehaving an unknown function, or in some aspects of the invention, encodea polypeptide having a known function. Preferably, a critical codingsequence encodes a polypeptide having an unknown function.

[0037] Preferably, a critical coding sequence is an essential codingsequence. An “essential coding sequence,” as used herein, is a codingsequence that encodes a polypeptide that is essential for the bacterialcell, preferably S. epidermidis, S. saprophyticus, or S. aureus, morepreferably, an S. aureus cell, to grow in vitro or in vivo, preferablyin vitro. Such polypeptides arc referred to herein as “essentialpolypeptides.” An essential coding sequence may encode a polypeptidehaving an unknown function, or in some aspects of the invention, encodea polypeptide having a known function. Preferably, an essential codingsequence encodes a polypeptide having an unknown function.

[0038] Identification of these critical coding sequences, preferablyessential coding sequences, provides a means for discovering new agentswith different targets and mechanisms of action compared to existingagents that are used to inhibit bacteria, preferably S. epidermidis, S.saprophyticus, or S. aureus, more preferably S. aureus. Theidentification of essential coding sequences of microbes, preferably S.epidermidis, S. saprophyticus, or S. aureus, more preferably S. aureus,that are useful in the present invention can begin by identifying codingsequences encoding a polypeptide, preferably, a polypeptide having noknown function. The coding sequences can be identified in databases,including, for instance, the S. aureus databases available from theUniversity of Oklahoma, TIGR, NCBI, Sanger, the HGS contig database, andthe HGS GSTS database. The identification of such coding sequences caninclude constructing contigs from data present in such databases.

[0039] As described herein, unknown coding sequences were typicallyidentified by analyzing publicly known polynucleotide sequences. Thedata obtained from the database contained the nucleotide sequence ofgenomic clones and predicted open reading frames. However, even thoughthe putative coding sequences may have been known, there was noindication that the coding sequences were in fact expressed, or in factcritical coding sequences. For instance, there is limited data known tothe art regarding regulatory regions required for the transcription of anucleotide sequence in S. aureus. Moreover, there is generally noevidence that the critical coding sequences and essential codingsequences identified herein are actually expressed. Thus, a person ofordinary skill, having the polynucleotide sequence of a genomic clone,would not be able to predict that an open reading frame would betranscribed, or that a coding sequence was critical, preferably,essential.

[0040] Typically, whether a coding sequence is a critical codingsequence, preferably, an essential coding sequence, can be determined byinactivating the coding sequence in a bacterial cell and determining thegrowth rate of the bacterial cell. Growth can be measured in vitro or invivo, preferably in vitro. Inactivating a coding sequence is done bymutating a coding sequence present in a bacterial cell. Mutationsinclude, for instance, a deletion mutation (i.e., the deletion ofnucleotides from the coding sequence), an insertion mutation (i.e., theinsertion of additional nucleotides into the coding sequence), anonsense mutation (i.e., changing a nucleotide of a codon so the codonencodes a different amino acid), and a missense mutation (i.e., changinga nucleotide of a codon so the codon functions as a stop codon). Someinsertion mutations and some deletion mutations result in frame-shiftmutations. Preferably, a coding sequence in a bacterial cell isengineered to contain a deletion.

[0041] In general, an internal fragment of a selected coding sequencecan be isolated or synthesized by methods known in the art, including,for instance, the polymerase chain reaction (PCR). Typically, theinternal fragment is about 150 base pairs to about 350 base pairs inlength, preferably about 300 base pairs. The internal fragmentpreferably corresponds to the 5′ end of the coding sequence. Preferably,the primers used to amplify the internal fragment contain a restrictionsite to allow ligation of the amplified internal fragment into a vector.For instance, when the vector is pSPT246 (described hereinbelow), oneprimer may contain a PstI site and the other primer may contain a SacIsite.

[0042] The internal fragment is typically ligated into a vector that canbe used to inactivate the coding sequence in the bacterial cell anddetermine if the coding sequence is a critical coding sequence or anessential coding sequence. Useful vectors include those that are unableto replicate under certain conditions in the bacterial cell thatcontains the coding sequence to be inactivated. Preferably, a vector isa temperature sensitive vector, i.e., it is unable to replicate in S.aureus at higher temperatures of, for instance, at least about 42° C.,or a vector is a suicide vector, i.e., it is unable to replicate in S.aureus. Preferably, a temperature sensitive vector is a shuttle vector,i.e., it is able to replicate in E. coli and S. aureus under theappropriate conditions. Examples of temperature sensitive plasmids thatcan be used to inactivate a coding sequence in S. aureus include pSPT181(Janzon and Arvidson, EMBO J., 9, 1391-1399 (1990)), and pSPT246(described hereinbelow). An example of a suicide plasmid that can beused to inactivate a coding sequence in S. aureus includes pKT4 (Tegmarket al., Mol. Microbiol., 37, 398-409 (2000)).

[0043] Using these methods, the following essential coding sequenceshave been identified: SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, and 137. Thepolypeptides encoded by the coding sequences are SEQ ID NOS: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 110, 114, 118, 122,126, 130, 134, and 138, respectively. Of these essential codingsequences, one coding sequence (SEQ ID NO: 33) encodes uridylate kinase,and is thus a known coding sequence. Prior to this invention theuridylate kinase coding sequence was not known to be essential for thegrowth of S. aureus. Using these methods, a critical coding sequencehaving the DNA sequence set forth at SEQ ID NO: 141 has been identified.The polypeptide encoded by the critical coding sequence is SEQ ID NO:142.

[0044] The coding sequences of the present invention include codingsequences that are similar to the coding sequences present in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 109, 113,117, 121, 125, 129, 133, 137, 141, or the complement thereof. Thesimilarity is referred to as structural similarity and is determined byaligning the residues of the two polynucleotides (i.e., the nucleotidesequence of the candidate coding sequence and the nucleotide sequence ofthe coding region of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137, 141, orthe complement thereof ) to optimize the number of identical nucleotidesalong the lengths of their sequences; gaps in either or both sequencesare permitted in making the alignment in order to optimize the number ofshared nucleotides, although the nucleotides in each sequence mustnonetheless remain in their proper order. A candidate coding region isthe coding region being compared to a coding region present in SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 109,113, 117, 121, 125, 129, 133, 137, or 141, or the complement thereof. Acandidate nucleotide sequence can be isolated from a microbe, preferablyS. aureus, or can be produced using recombinant techniques, orchemically or enzymatically synthesized. Preferably, two nucleotidesequences are compared using the Blastn program of the BLAST 2 searchalgorithm, as described by Tatusova, et al. (FEMS Microbiol Lett 1999,174:247-250), and available at www.ncbi.nlm.nih.gov/gorf/b12.html.Preferably, the default values for all BLAST 2 search parameters areused, including reward for match=1, penalty for mismatch=−2, open gappenalty=5, extension gap penalty=2, gap x_dropoff=50, expect=10,wordsize=11, and filter on. In the comparison of two nucleotidesequences using the BLAST search algorithm, structural similarity isreferred to as “identities.” Preferably, a polynucleotide includes anucleotide sequence having a structural similarity with the codingregion of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137, 141, or thecomplement thereof, of, in increasing order of preference, at leastabout 57%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, most preferably at least about 95% identity.

[0045] The present invention includes isolated polynucleotides thatinclude a nucleotide sequence SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137,141, or the complement thereof. As used herein, an “isolated”polypeptide or polynucleotide means a polypeptide or polynucleotide thathas been either removed from its natural environment, produced usingrecombinant techniques, or chemically or enzymatically synthesized.Preferably, a polypeptide or polynucleotide of this invention ispurified, i.e., essentially free from any other polypeptides orpolynucleotides and associated cellular products or other impurities. Anisolated polynucleotide of the invention may include a nucleotidesequence having, in increasing order of preference, at least about 57%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, most preferably at least about 95% structural similarity witha nucleotide sequence SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137, 141, orthe complement thereof, where the isolated polynucleotide includes acritical coding sequence, preferably, an essential coding sequence. Thepresent invention also includes the polypeptides encoded by the codingsequences.

[0046] Another aspect of the invention includes isolated polynucleotidesconsisting essentially of a nucleotide sequence SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121,125, 129, 133, 137, or 141, or the complement thereof. Thepolynucleotide optionally further includes from zero to up to about5,000 nucleotides upstream and/or downstream of the nucleotide sequenceSEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 109, 113, 117, 121, 125, 129, 133, 137, 141, or the complementthereof. An isolated polynucleotide of the invention may consistessentially of a nucleotide sequence having, in increasing order ofpreference, at least about 57%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, most preferably at least about95% structural similarity with a nucleotide sequence SEQ ID NO: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117,121, 125, 129, 133, 137, 141, or the complement thereof, where theisolated polynucleotide includes an essential coding sequence. Thepolynucleotide optionally further includes from zero to up to about5,000 nucleotides upstream and/or downstream of the nucleotide sequenceSEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 109, 113, 117, 121, 125, 129, 133, 137, 141,or the complementthereof. The present invention also includes the polypeptides encoded bythe coding sequences.

[0047] Insertional inactivation of critical coding sequences,preferably, essential coding sequences, allows different classes ofcoding sequences to be identified. Examples of different classesinclude, for instance, coding sequences encoding proteins involved incell surface metabolism, enzymes involved in cellular biosyntheticpathways including cell wall biosynthesis and assembly, components ofthe TCA cycle, proteins similar to oligopeptide transport proteins ofthe ATP-binding cassette (ABC) transporter superfamily, and involved incellular regulatory and repair processes, and coding sequences affectingmorphogenesis and cell division, secretion and sorting of proteins, andsignal transduction systems.

[0048] The critical coding sequences, preferably, essential codingsequences may be cloned by PCR, using microbial, preferably S.epidermidis, S. saprophyticus, or S. aureus, more preferably S. aureus,genomic DNA as the template. For ease of inserting the open readingframe into expression vectors, PCR primers may be chosen so that thePCR-amplified coding sequence has a restriction enzyme site at the 5′end preceding the initiation codon ATG, and a restriction enzyme site atthe 3′ end after the termination codon TAG, TGA or TAA. If desirable,the codons in the coding sequence may be changed, without changing theamino acids, to optimize expression of a polypeptide encoded by anessential coding sequence. For instance, if an essential coding sequenceis to be expressed in E. coli, the codons of the coding sequence can bechanged to comply with the E. coli codon preference (see, for instance,Grosjean and Fiers, Gene, 18, 199-209 (1982), and Konigsberg et al.,Proc. Natl. Acad. Sci., USA, 80, 687-691 (1983)). Optimization of codonusage may lead to an increase in the expression of the encodedpolypeptide when produced in a microbe other than the microbe from whichthe essential coding sequence was isolated. If the polypeptide is to beproduced extracellularly, either in the periplasm of, for instance, E.coli or other bacteria, or into the cell culture medium, the codingsequence may be cloned without its initiation codon and placed into anexpression vector behind a signal sequence.

[0049] Proteins may be produced in prokaryotic or eukaryotic expressionsystems using known promoters, vectors, and hosts. Such expressionsystems, promoters, vectors, and hosts are known to the art. A suitablehost cell may be used for expression of the polypeptide, such as E.coli, other bacteria, including Bacillus and S. aureus, yeast, includingPichia pastoris and Saccharomyces cerevisiae, insect cells, or mammaliancells, including CHO cells, using suitable vectors known in the art.Proteins may be produced directly or fused to a polypeptide, and eitherintracellularly or extracellularly by secretion into the periplasmicspace of a bacterial cell or into the cell culture medium. Secretion ofa protein typically requires a signal peptide (also known aspre-sequence); a number of signal sequences from prokaryotes andeukaryotes are known to function for the secretion of recombinantproteins. During the protein secretion process, the signal peptide isremoved by signal peptidase to yield the mature protein.

[0050] The polypeptide encoded by a critical coding sequence,preferably, an essential coding sequence, may be isolated. To simplifythe isolation process, a purification tag may be added either at the 5′or 3′ end of the coding sequence. Commonly used purification tagsinclude a stretch of six histidine residues (U.S. Pat. Nos. 5,284,933and 5,310,663), a streptavidin-affinity tag described by Schmidt andSkerra, Protein Engineering, 6, 109-122 (1993), a FLAG peptide (Hopp etal, Biotechnology, 6, 1205-1210 (1988)), glutathione S-transferase(Smith and Johnson, Gene, 67, 31-40 (1988)), and thioredoxin (LaVallieet al., Bio/Technology, 11, 187-193 (1993)). To remove these tags, aproteolytic cleavage recognition site may be inserted at the fusionjunction. Commonly used proteases are factor Xa, thrombin, andenterokinase. Preferably, a polypeptide encoded by an essential codingsequence is isolated, more preferably, purified.

[0051] The identification of critical coding sequences, preferably,essential coding sequences, renders them useful in methods ofidentifying new agents according to the present invention. Such methodsinclude assaying potential agents for the ability to interfere withexpression of a critical coding sequence, preferably, an essentialcoding sequence, thereby preventing the expression and decreasing theconcentration of a polypeptide encoded by the coding sequence. Withoutintending to be limiting, it is anticipated that agents can act by, forinstance, interacting with a critical coding sequence, preferably, anessential coding sequence, interacting with a nucleotide sequence thatis adjacent to a critical coding sequence, preferably, an essentialcoding sequence (e g., a promoter sequence), or inhibiting expression ofa polypeptide involved in regulating expression of a critical codingsequence, preferably, an essential coding region. Agents that can beused to inhibit the expression of a critical coding sequence,preferably, an essential coding region include, for instance, the use ofanti-sense polynucleotides that are complementary to the mRNA moleculestranscribed from the coding sequence, and double stranded RNA (Fire etal., Nature, 391, 806-11 (1998)).

[0052] Such methods also include assaying potential agents for theability to bind to a polypeptide encoded in whole or in part by a DNAsequence set forth in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133,137, 141 or the complementary strand thereof. Optionally, agents thatbind to such a polypeptide can be further evaluated to determine if theyinhibit the function of the polypeptide to which they bind.

[0053] A polypeptide produced by a critical coding sequence, preferably,an essential coding sequence, may be used in assays including, forinstance, high throughput assays, to screen for agents that inhibit thefunction of the polypeptide. The sources for potential agents to bescreened include. for instance, chemical compound libraries,fermentation media of Streptomycetes, other bacteria and fungi, and cellextracts of plants and other vegetations. For proteins with knownenzymatic activity, assays may be established based on the activity, anda large number of potential agents can be screened for ability toinhibit the activity. Such assays are referred to herein as “enzymeassays.” Enzyme assays vary depending on the enzyme, and typically areknown to the art.

[0054] For proteins that interact with another protein or nucleic acid,assays can be established to measure such interaction directly, and thepotential agents screened for the ability to inhibit the bindinginteraction (referred to herein as “binding assays”). In another aspectof the invention, assays can be established allowing the identificationof agents that bind to a polypeptide encoded by an essential codingsequence (referred to herein as “ligand binding assays”).

[0055] For proteins that interact with another protein or nucleic acid,such binding interactions may be evaluated indirectly using the yeasttwo-hybrid system described in Fields and Song (Nature, 340, 245-246(1989)), and Fields and Sternglanz (Trends in Genetics, 10, 286-292(1994)). The two-hybrid system is a genetic assay for detectinginteractions between two polypeptides. It can be used to identifyproteins that bind to a known protein of interest, or to delineatedomains or residues critical for an interaction. Variations on thismethodology have been developed to clone coding sequences that encodeDNA-binding proteins, to identify polypeptides that bind to a protein,and to screen for drugs. The two-hybrid system exploits the ability of apair of interacting proteins to bring a transcription activation domaininto close proximity with a DNA-binding domain that binds to an upstreamactivation sequence (UAS) of a reporter coding sequence, and isgenerally performed in yeast. The assay requires the construction of twohybrid coding sequences encoding (1) a DNA-binding domain that is fusedto a protein X, and (2) an activation domain fused to a protein Y. TheDNA-binding domain targets the first hybrid protein to the UAS of thereporter coding sequence; however, because most proteins lack anactivation domain, this DNA-binding hybrid protein does not activatetranscription of the reporter coding sequence. The second hybridprotein, which contains the activation domain, cannot by itself activateexpression of the reporter because it does not bind the UAS. However,when both hybrid proteins are present, the noncovalent interaction ofprotein X and protein Y tethers the activation domain to the UAS,activating transcription of the reporter coding sequence. When thepolypeptide encoded by, for instance, an essential coding sequence(protein X, for example) is already known to interact with anotherprotein or nucleic acid (protein Y, for example), this binding assay canbe used to detect agents that interfere with the interaction of X and Y.Expression of the reporter coding sequence is monitored as differenttest agents are added to the system; the presence of an inhibitory agentinhibits binding and results in lack of a reporter signal.

[0056] When the function of a polypeptide encoded by, for instance., anessential coding sequence is unknown and no ligands are known to bindthe polypeptide, the yeast two-hybrid assay can also be used to identifyproteins that bind to the polypeptide. In an assay to identify proteinsthat bind to protein X (the target protein), a large number of hybridcoding sequences, each containing a different protein Y, are producedand screened in the assay. Typically, Y is encoded by a pool of plasmidsin which total cDNA or genomic DNA is ligated to the activation domain.This system is applicable to a wide variety of proteins, and it is noteven necessary to know the identity or function of protein Y. The systemis highly sensitive and can detect interactions not revealed by othermethods; even transient interactions may trigger transcription toproduce a stable mRNA that can be repeatedly translated to yield thereporter protein. When a protein is identified that binds to anessential polypeptide, the two-hybrid system can be used in a bindingassay to identify agents that inhibit binding and result in lack of areporter signal.

[0057] Ligand binding assays known to the art may be used to search foragents that bind to the target protein. Without intending to belimiting, one such screening method to identify direct binding of testligands to a target protein is described in Bowie et al. (U.S. Pat. No.5,585,277). This method relies on the principle that proteins generallyexist as a mixture of folded and unfolded states, and continuallyalternate between the two states. When a test ligand binds to the foldedform of a target protein (i.e., when the test ligand is a ligand of thetarget protein), the target protein molecule bound by the ligand remainsin its folded state. Thus, the folded target protein is present to agreater extent in the presence of a test ligand which binds the targetprotein. than in the absence of a ligand. Binding of the ligand to thetarget protein can be determined by any method which distinguishesbetween the folded and unfolded state of the target protein. Thefunction of the target protein need not be known in order for this assayto be performed.

[0058] Another method for identifying ligands for a target protein isdescribed in Wieboldt et al., Anal Chem., 69, 1683-1691 (1997). Thistechnique screens combinatorial libraries of 20-30 agents at a time insolution phase for binding to the target protein. Agents that bind tothe target protein are separated from other library components bycentrifugal ultrafiltration. The specifically selected molecules thatare retained on the filter are subsequently liberated from the targetprotein and analyzed by HPLC and pneumatically assisted electrospray(ion spray) ionization mass spectroscopy. This procedure selects librarycomponents with the greatest affinity for the target protein, and isparticularly useful for small molecule libraries.

[0059] Another method allows the identification of ligands present in asample using capillary electrophoresis (Hughes et al., U.S. Pat. No.5,783,397). The sample and the target protein are combined and resolved.The conditions of electrophoresis results in simultaneouslyfractionating the components present in the sample and screening forcomponents that bind to the target molecule. This method is particularlyuseful for complex samples including, for instance, extracts of plants,animals, microbes, or portions thereof and chemical libraries producedby, for instance, combinatorial chemistry.

[0060] The agents identified by the initial screens are evaluated fortheir effect on survival of microbes, preferably S. epidermidis, S.saprophyticus, or S. aureus, more preferably S. aureus. Agents thatinterfere with bacterial survival are expected to be capable ofpreventing the establishment of an infection or reversing the outcome ofan infection once it is established. Agents may be bacteriocidal (i.e.,an agent kills the microbe and prevents the replication of the microbe)or bacteriostatic (i.e., an agent reversibly prevents replication of themicrobe). Preferably, the agent is bacteriocidal. Such agents will beuseful to treat a subject infected with S. epidermidis, S.saprophyticus, or S. aureus, preferably S. aureus, or at risk of beinginfected by S. epidermidis, S. saprophyticus, or S. aureus, preferablyS. aureus.

[0061] The identification of S. aureus critical coding sequences,preferably, essential coding sequences, also provides for microorganismsexhibiting reduced virulence, which are useful in vaccines. The term“vaccine” refers to a composition that, upon administration to asubject, will provide protection against S. epidermidis, S.saprophyticus, or S. aureus, preferably, S. aureus. Administration of avaccine to a subject will produce an immunological response to the S.aureus and result in immunity. A vaccine is administered in an amounteffective to result in some therapeutic benefit or effect so as toresult in an immune response that inhibits or prevents an infection byS. aureus in a subject, or so as to result in the production ofantibodies to an S. aureus.

[0062] Such microorganisms that can be used in a vaccine include S.epidermidis, S. saprophyticus, or S. aureus, preferably S. aureus,mutants containing a mutation in a coding sequence represented by anyone of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 109, 113, 117, 121, 125, 129, 133, 137, 141, or a codingsequence having structural similarity thereto. Optionally, an S.epidermidis, S. saprophyticus, or S. aureus, preferably an S. aureus,includes more than one mutation. The reduced virulence of theseorganisms and their immunogenicity may be confirmed by administration toa subject. Animal models useful for evaluating S. aureus virulence in avariety of conditions, including for example, pneumonia, peritonitis,endophthalmitis, endocarditis, septicemia, and arthritis, are known tothe art.

[0063] While it is possible for an avirulent microorganism of theinvention to be administered alone, one or more of such mutantmicroorganisms are preferably administered in a vaccine compositioncontaining a suitable adjuvant(s) and a pharmaceutically acceptablediluent(s) or carrier(s). The carrier(s) must be “acceptable” in thesense of being compatible with the avirulent microorganism of theinvention and not deleterious to the subject to be immunized. Typically,the carriers will be water or saline which will be sterile and pyrogenfree. The subject to be immunized is a subject needing protection from adisease caused by a virulent form of S. aureus.

[0064] Any adjuvant known in the art may be used in the vaccinecomposition, including oil-based adjuvants such as Freund's CompleteAdjuvant and Freund's Incomplete Adjuvant, mycolate-based adjuvants(e.g., trehalose dimycolate), bacteria lipopolysaccharide (LPS),peptidoglycans (i.e., mumins, mucopeptides, or glycoproteins such asN-Opaca, muramyl dipeptide (MDP), or MDP analogs), proteoglycans (e.g.,extracted from Klebsiela spp.), streptococcal preparations (e.g.,OK432), the “Iscoms” of EP 109 942, EP 180 564 and EP 231 039, aluminumhydroxide, saponin, DEAE-dextran, neutral oils (such as miglyol),vegetable oils (such as arachis oil), liposomes, the Ribi adjuvantsystem (see, for example GB-A-2 189 141), or adjuvants available underthe trade designation BIOSTIM (e.g., 01K2) and PLURONIC polyols.Recently, an alternative adjuvant consisting of extracts of Amycolata, abacterial genus in the order Actinomycetales, has been described in U.S.Pat. No. 4,877,612. Additionally, proprietary adjuvant mixtures arecommercially available. The adjuvant used will depend, in part, on therecipient organism. The amount of adjuvant to administer will depend onthe type and size of animal. Optimal dosages may be readily determinedby routine methods.

[0065] The vaccine compositions optionally may include pharmaceuticallyacceptable (i.e., sterile and non-toxic) liquid, semisolid, or soliddiluents that serve as pharmaceutical vehicles, excipients, or media.Any diluent known in the art may be used. Exemplary diluents include,but are not limited to, polyoxyethylene sorbitan monolaurate, magnesiumstearate, methyl-and propylhydroxybenzoate, talc, alginates, starches,lactose, sucrose, dextrose, sorbitol, mannitol, gum acacia, calciumphosphate, mineral oil, cocoa butter, and oil of theobroma.

[0066] The vaccine compositions can be packaged in forms convenient fordelivery. The compositions can be enclosed within a capsule, sachet,cachet, gelatin, paper or other container. These delivery forms arepreferred when compatible with entry of the immunogenic composition intothe recipient organism and, particularly, when the immunogeniccomposition is being delivered in unit dose form. The dosage units canbe packaged, e.g., in tablets, capsules, suppositories or cachets.

[0067] The vaccine compositions may be introduced into the subject to beimmunized by any conventional method including, e.g., by intravenous,intradermal, intramuscular, intramammary, intraperitoneal, orsubcutaneous injection; by oral, sublingual, nasal, anal, vaginal, ortransdermal delivery; or by surgical implantation, e.g., embedded underthe splenic capsule or in the cornea. The treatment may consist of asingle dose or a plurality of doses over a period of time.

[0068] It will be appreciated that the vaccine of the invention may beuseful in the fields of human medicine and veterinary medicine. Thus,the subject to be immunized may be a human or an animal, for example,cows, sheep., pigs, horses, dogs, cats, and poultry such as chickens,turkeys, ducks and geese.

[0069] The present invention is illustrated by the following examples.It is to be understood that the particular examples, materials, amounts,and procedures are to be interpreted broadly in accordance with thescope and spirit of the invention as set forth herein.

EXAMPLE 1 Identification of Critical and Essential S. aureus CodingSequences

[0070] Identification of Unknown Coding Sequences

[0071] There are about 3500 open reading frames in the HGS database ofS. aureus nucleotide sequences. A Fast A homology search was conductedon these open reading frames. This homology search of those open readingframes indicated that 662 of the open reading frames were unknown codingsequences. The methods described herein typically require an openreading frame of about 300 base pairs; 492 of the 662 open readingframes were at least 300 base pairs. Of these 492, 60 had homology withunknown open reading frames from other bacterial species, 270 had nohomology with any open reading frames, and 160 had homology witheukaryotic coding sequences. A homology search was also conductedbetween the predicted coding sequences of the Mycoplasma genitaliumgenome and the coding sequences of S. aureus.

[0072] The nucleotide sequences of the unknown coding sequences areshown in Table 1. Whether these coding sequences were critical oressential was determined as described herein. TABLE 1 Primers used toamplify unknown coding sequences from S. aureus Nucleotide sequence ofPrimer pair used to Predicted unknown coding sequence amplify codingsequence polypeptide SEQ ID NO:1 SEQ ID NOs:35-36 SEQ ID NO:2 SEQ IDNO:3 SEQ ID NOs:37-38 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NOs:39-40 SEQ IDNO:6 SEQ ID NO:7 SEQ ID NOs:41-42 SEQ ID NO:8 SEQ ID NO:9 SEQ IDNOs:43-44 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NOs:45-46 SEQ ID NO:12 SEQ IDNO:13 SEQ ID NOs:47-48 SEQ ID NO:14 SEQ ID NO:15 SEQ ID NOs:49-50 SEQ IDNO:16 SEQ ID NO:17 SEQ ID NOs:51-52 SEQ ID NO:18 SEQ ID NO:19 SEQ IDNOs:53-54 SEQ ID NO:20 SEQ ID NO:21 SEQ ID NOs:55-56 SEQ ID NO:22 SEQ IDNO:23 SEQ ID NOs:57-58 SEQ ID NO:24 SEQ ID NO:25 SEQ ID NOs:59-60 SEQ IDNO:26 SEQ ID NO:27 SEQ ID NOs:61-62 SEQ ID NO:28 SEQ ID NO:29 SEQ IDNOs:63-64 SEQ ID NO:30 SEQ ID NO:31 SEQ ID NOs:65-66 SEQ ID NO:32 SEQ IDNO:33 SEQ ID NOs:67-68 SEQ ID NO:34 SEQ ID NO:109 SEQ ID NOs:111-112 SEQID NO:110 SEQ ID NO:113 SEQ ID NOs:115-116 SEQ ID NO:114 SEQ ID NO:117SEQ ID NOs:119-120 SEQ ID NO:118 SEQ ID NO:121 SEQ ID NOs:123-124 SEQ IDNO:122 SEQ ID NO:125 SEQ ID NOs:127-128 SEQ ID NO:126 SEQ ID NO:129 SEQID NOs:131-132 SEQ ID NO:130 SEQ ID NO:133 SEQ ID NOs:135-136 SEQ IDNO:134 SEQ ID NO:137 SEQ ID NOs:139-140 SEQ ID NO:138 SEQ ID NO:141 SEQID NOs:143-144 SEQ ID NO:142

[0073] Insertion Inactivation of Unknown Coding Sequences

[0074] Inactivation was achieved by integration of a plasmid in the 5′half of the target coding sequence by homologous recombination. Aninternal fragment of the selected coding sequence was synthesized by PCR. The length of the amplified fragment was between about 250 base pairsto about 350 base pairs, and included the 5′ end of the coding sequence.The primers used for amplification included additional nucleotides suchthat a PstI restriction site was added to one end of the amplifiedfragment and a SacI restriction site was added to the other end of theamplified fragment. The primers are shown in Table 1. The addedrestriction sites allowed ligation of the amplified fragment to thetemperature sensitive shuttle vector pSPT246. pSPT264 was constructed byligating pRN8103 and pSP64-PolyA. The pRN8103 thermosensitivereplication vector contains a unique EcoRI restriction site and thevector cannot replicate in E. coli. pRN8103 is described in Novick etal., (J. Mol. Biol., 192, 209-220 (1986)). The pSP64-PolyA vector,obtained from Promega Corp. (Madison, Wis.), replicates in E. coli, butnot in S. aureus. pSP64-PolyA also contains a unique EcoRI restrictionsite. An E. coli/S. aureus shuttle vector was constructed by digestingeach vector with EcoRI, ligating the two vectors together, andtransforming the DNA into E. coli. The resulting shuttle vector wasdesignated pSPT264.

[0075] The recombinant plasmid (i.e., pSPT246 containing an amplifiedfragment) was used to transform E. coli, isolated, and then transferredto S. aureus RN4220 (described in Kreiswirth et al., Nature, 305,709-712 (1983)) by electroporation. Transformants were selected byincubation on Nutrient agar plates containing tetracycline (10 μg/ml) atthe permissive temperature (30° C.). The presence of the correct plasmidwas verified by PCR.

[0076] One clone with the correct plasmid was grown on Nutrient agarwith tetracycline (10 μg/ml) at 32° C. overnight to allow recombinationbetween the plasmid and the selected chromosomal allele. To select forrecombinants the bacteria were then grown at the non-permissivetemperature (43° C.) for 18 hours in Brain Heart Infusion (BHI) brothwithout tetracycline, followed by a 1:10 dilution into BHI brothcontaining 5 μg/ml tetracycline. The cells were incubated overnight at43° C. The bacterial culture was then diluted, spread on Nutrient agarplates containing 5 μg/ml tetracycline and incubated at 43° C.overnight. As the plasmid cannot replicate at 43° C., only cells withthe plasmid integrated into the chromosome are tetracycline resistantand form colonies. Micro-colonies that appear at the non-permissivetemperature are also considered, as they may represent mutations incoding sequences that are important (e.g., critical), but not essential,for growth.

[0077] The plasmid integrates at a low frequency at other sites in thechromosome, thus tetracycline resistant clones appeared even when thetarget coding sequence was essential. Therefore, ten colonies from eachselection at 43° C. were tested for specific integration of the plasmidinto the selected target coding sequence by PCR. A primer pairconsisting of one primer that binds to the vector DNA, and a secondprimer that binds upstream of the target coding sequence in thechromosome was used for PCR amplification. The primer pair amplifies theintervening chromosomal-vector region, and an amplified DNA fragment isproduced only if the vector integrated at the predicted location. Theabsence of a band suggests the vector cannot integrate, and that thecoding sequence is essential. Typically, all or none out of the testedcolonies were specific recombinants. In those cases where norecombinants are found the target coding sequence is consideredessential. For a number of target coding sequences (both essential andnon-essential) the same results have been obtained when the wholeselection procedure was repeated.

[0078] This protocol has successfully been used to analyze about 300 outof the of 492 unknown complete or partial coding sequences identified.Out of the analyzed coding sequences, 26 appeared to be critical andwere further analyzed as described below.

EXAMPLE 2 Cloning of Essential S. aureus Coding Sequences and Expressionin E. coli

[0079] Overview of the Expression System and Cloning Procedure

[0080] The overexpression of S. aureus proteins is accomplished usingthe Qiagen Type ATG expression system (Qiagen Gmbh, Santa Clara,Calif.). This system utilizes E. coli strain “M15” whose genotype hasbeen described by Qiagen as Nal^(s), Str^(s), rif^(s), lac⁻, ara⁻, gal⁻,mtl⁻, F⁻, recA⁺, uvr⁺. Two replication compatible vectors, pREP4 andpQE-60 (each obtained from Qiagen), are introduced into the M15 strainduring the procedure. Alternatively, pQE-70 can be used instead ofpQE-60. The pREP4 vector is a pACYC-derived vector that contains the ladgene encoding for the Lactose (LacI repressor protein, and the vectorencodes kanamycin drug resistance. The expression vector pQE-60 is apBR322-derived vector that contains a modified T5 phage promoter, astrong ribosome binding site (RBS), and the coding sequence of thespecific S. aureus coding sequence to be expressed. The T5 promotermodifications include the placement of operator sites for binding andregulation of the promoter by the LacI repressor. Induction ofexpression is performed by the addition of IPTG(isopropylthio-β-D-galactoside) to a log phase culture.

[0081] The general cloning strategy is to first amplify the specificcoding sequence from S. aureus genomic DNA using PCR primers to the 5′and 3′ ends of the coding sequence sequence. The PCR primers aredesigned to add an NcoI and a BglII restriction site at the 5′ and 3′ends of the coding sequence respectively. The coding sequence should befree of any NcoI or BglII restriction sites. If such sites are present,they are eliminated using site-directed PCR mutagenesis procedures knownto the art. Alternatively, a different restriction site, for instance aBamHI restriction site, is used instead of a BglII restriction site. Theamplified S. aureus coding sequence is ligated into pCR-2. 1(Invitrogen, Carlsbad, Calif.) and transformed into E. coli usingtechniques known to the art. Colonies are screened for the presence ofthe coding sequence by PCR amplification or vector restriction analysis.Clones are randomly selected and the nucleotide sequence of the insertDNA, i.e., the S. aureus coding sequence, is determined to confirmauthenticity of the insert.

[0082] The pCR-2.1 vector containing the desired coding sequence isdigested with NcoI/BglII and the coding sequence is isolated and ligatedinto the corresponding NcoI/BglII restriction sites of pQE-60. Theligation mixture is used to electroporate the vector DNA into the M15strain that contained the pREP4 vector. The resulting transformants arescreened by PCR or restriction analysis. Candidates are grown in ashake-flask and screened for the over-expression of a protein bandhaving the appropriate size as analyzed by SDS-PAGE or Western analysis.Anti-His antibody (Invitrogen) is used in the Western analysis. A singlecandidate is selected for the overexpresion and isolation of the proteinencoded by each coding sequence.

[0083] Culture and Media

[0084] The medium for cloning and maintenance of cells containingrecombinant plasmids in E. coli is LB supplemented with the appropriateantibiotic (100 μg/ml ampicillin, 25 μg/ml kanamycin). S. aureus wasgrown in Mueller-Hinton medium. Competent INVF′α cells (Invitrogen,Carlsbad, Calif.) are used according to the manufacturer's direction.The M15 pREP-4 strain was purchased from Qiagen. SOC medium was used inthe electroporation of cells. LB and SOC media are described in Sambrooket al. (Molecular Cloning: A Laboratory Manual., Cold Spring HarborLaboratory Press, pp. A1-A4 (1989)). Mueller-Hinton medium is describedin Atlas et al., Handbook of Microbiological Media, CRC Press.

[0085] Design of the pQE60 Expression Vector

[0086] The portion of the pQE-60 DNA sequence containing the T5promoter, the RBS, the ATG start codon (in bold), the NcoI restrictionsite (underlined), the BglII restriction site (underlined), 6 His tag(double underline), and the TAA stop codon (in bold) is shown (SEQ IDNO: 89): CTCGAGAAAT CATAAAAAAT TTATTTGCTT TGTGAGCGGA TAACAATTATAATAGATTCA ATTGTGAGCG GATAACAATT TCACACAGAA TTCATTAAAG AGGAGAAATTAACCATGGGA GGATCCAGAT CT CATCACCA TCACCATCAC TAAGCTTAAT TA NcoI        BglII

[0087] The S. aureus coding sequences are modified by PCR to containcompatible in-frame NcoI and BglII restriction sites.

[0088] Primer Design

[0089] The general formula for the design of the primer to the 5′portion of the S. aureus coding sequence is usually 5′- CCATGGGAN₂₀₋₃₀,and the general formula for the 3′ primer is usually 5′- AGATCTN₂₀₋₃₀.These primers add the NcoI and BglII restriction sequences. The first“N” nucleotide of the 5′ sequence correspond to the codon of the secondamino acid of the S. aureus coding sequence after its ATG start. Thefirst “N” nucleotide of the 3′ primer corresponds to the thirdnucleotide in the codon preceding the stop codon of the S. aureus codingsequence. The number of nucleotides to include in the primer varieddepending on the specific DNA sequence, but is typically in a range of20 to 30 bases. The primers are phosphorylated. Examples of primers thatcan be used to amplify some coding sequences are shown in Table 2. TABLE2 Primers used to amplify essential coding sequences from S. aureusResulting Predicted Essential Primer pair used to sequence inpolypeptide coding clone coding pQE-60 or and predicted sequencesequence pQE-70 molecular weight SEQ ID NO:1 SEQ ID NOs:91-92 SEQ IDNO:69 SEQ ID NO:70. 28.1 kD SEQ ID NO:3 SEQ ID NOs:93-94 SEQ ID NO:71SEQ ID NO:72, 31.6 kD SEQ ID NO:5 SEQ ID NOs:95-96 SEQ ID NO:73 SEQ IDNO:74, 28.5 kD SEQ ID NO:9 SEQ ID NOs:97-98 SEQ ID NO:75 SEQ ID NO:76,37.4 kD SEQ ID NO:11 SEQ ID NOs:99- SEQ ID NO:77 SEQ ID NO:78, 100 40.9kD SEQ ID NO:13 SEQ ID NOs:101- SEQ ID NO:79 SEQ ID NO:80, 102 30.4 kDSEQ ID NO:15 SEQ ID NOs:103- SEQ ID NO:81 SEQ ID NO:82, 104 78.1 kD SEQID NO:17 SEQ ID NOs:105- SEQ ID NO:83 SEQ ID NO:84, 106 57.4 kD SEQ IDNO:19 SEQ ID NOs:107- SEQ ID NO:85 SEQ ID NO:86, 108 16.1 kD

[0090] Preparation of the S. aureus Genomic DNA

[0091] Strain 1SP3 (obtained from S. Arvidson, Karolinska Institute) isused to inoculate 10 mls of Mueller-Hinton broth. After overnight growthal 37° C., 1.5 mls of culture are pelleted in an eppendorf tube and thenresuspended in 400 μl of TE, pH 8.0 (Sambrook et al. (Molecular Cloning:A Laboratory Manual., Cold Spring Harbor Laboratory Press, p. B.20(1989)). Following the addition of 50 μl lysostaphin solution (10mg/ml), the cells are incubated at 37° C. for 1 hour. Seventymicroliters of 10% SDS and 10 μl of proteinase K (20 mg/ml) are addedand the incubation continued at 37° C. for another hour. After theaddition of 100 μl of 5 M NaCl, the cell suspension is vortexed and 80μl of a solution containing 10% hexadecyltrimethyl ammonium bromide, 0.7M NaCl (CTAB/NaCl) is added. The cells are vortexed and then incubatedat 65° C. for 10 minutes. Following the addition of an equal volume of25:24:1 phenol:chloroform:isoamyl alcohol, the cells are vortexed andcentrifuged for 5 minutes. The aqueous phase is then transferred to afresh tube, leaving behind the white CTAB/NaCl interface. The extractionis repeated, and the aqueous layer is again transferred to a fresh tube.Following the addition of an equal volume of isopropanol, the tube isgently mixed causing a stringy precipitate to form. A Pasteur pipettefashioned into a small hook is used to gently remove the precipitate andto transfer it into another tube containing 1 ml of 70% ethanol. Thetube is centrifuged, and the resulting pellet is washed once with 70%ethanol. After drying, the DNA pellet is resuspended in 100 μl of waterand the concentration of the recovered DNA is determined usingtechniques known in the art.

[0092] PCR Amplification

[0093] PCR reactions are performed using either the Perkin-Elmer CetusGeneAmp 9600 or 2400 thermal cyclers (Perkin-Elmer, Norwalk, Conn.). Thedeoxynucleotide mix and the Pfu DNA polymerase are purchased fromStratagene (La Jolla, Calif.). The AmpliTaq Gold kit is purchased fromPerkin Elmer. The PCR synthesis protocol for long template amplificationis as follows: 1 μg of S. aureus genomic DNA, 10 μl of 10× reactionbuffer (with 15 mM MgCl₂), 500 ng of each primer, 16 μl of 1.25 mMdNTP's, 1 μl of AmpliTaq Gold, and water to 100 μl are added per PCRmicrotube. The DNA is amplified for 35 cycles using Cycle Program of 95°C. for 5 minutes followed by 35 cycles of 94° C. for 30 seconds, 50° C.for 1 minute and 72° C. for 3 minutes, an extension at 72° C. for 5minutes, and finally 40° C. on hold. A 10 μl aliquot of the synthesisreaction is loaded onto a 1.2% agarose gel to confirm the presence andsize of the synthesized fragment. The PCR product is produced bycombining multiple PCR reaction, EtOH precipitating the DNA, and cuttingthe desired fragment out of a 1.2% agarose gel. The DNA is isolated fromthe agarose using Amicon Ultrafree-DA extraction filters (MilliporeCorp., Bedford, Mass.). The filters are used according to manufacturer'sdirections.

[0094] Ligation and Transformation

[0095] The pQE-60 vector and the pCR2.1 vector containing the S. aureuscoding sequence are digested with NcoI and BglII restriction enzymes.The pQE-60 vector fragment and the S. aureus coding sequence areisolated from an agarose gel. The two DNAs are ligated and transformedinto electrocompetent M15 cells containing pREP-4, and plated on LB agarwith ampicillin and kanamycin supplementation. Ligase is purchase fromBioLab (Beverley, Mass.), and used in accordance with the manufacturer'sinstructions. Electroporation of the ligated DNA into M15 pREP-4 cellsis performed using a Bio-Rad Gene Pulser (Hercules, Calif.). Competentcells are prepared from 1 liter of cells with an optical density of 1 atA₅₅₀. The cells are chilled and washed successively with 1 liter and 0.5liters of ice cold sterile water. The cells are resuspended in 20 mls ofice cold sterile 10% glycerol, re-centrifuged and placed into a finalsuspension of 2 to 3 mls of cold sterile 10% glycerol. Fifty microlitersof cells are mixed with 5 μls or less of ligated DNA. The cell/DNAmixture is transferred to an electroporation cuvette and pulsed with thesettings at 25 μF, 2.5 kV, and the Pulse Controller set to 200 Ω. One mlof SOC media is then added. The cells are incubated at 30° C. for onehour and plated on selective media.

[0096] Several resultant colonies from the transformation are selectedat random and vector DNA is isolated using the Miniprep or Maxiprep kitspurchased from Qiagen. The vector DNA is isolated according to themanufacturer's instructions. The candidates are screened by restrictionenzyme digestions. Restriction enzymes are purchased from New EnglandBioLab (Beverly, Mass.). Restriction enzymes are used according to themanufacturer's instructions.

[0097] Expression Conditions

[0098] The expression culture is streaked on an LB plate containingampicillin and kanamycin. A single colony isolate is used to inoculate50 mls of LB medium supplemented with ampicillin and kanamycin and grownovernight at the desired temperature. Following sub-culture into thesuitable volume of the identical media at 0.50 A₅₅₀/ml, the culture isgrown at the same temperature with vigorous aeration until an A₅₅₀ of3.0 was reached. The culture is induced by the addition of IPTG to afinal concentration of 1 mM. Culture aliquots are removed at 0, 2, and 4hours post-induction for SDS-PAGE or Western analysis. Cells areharvested for protein isolation between 4 and 6 hours. Proteins areisolated using a metal-chelate affinity chromatography purificationsystem (QIAEXPRESS, Qiagen).

EXAMPLE 3 Use of Essential Coding Sequence Products in Screen forAntimicrobial Agents

[0099] Individual purified proteins (i.e., target proteins) are combinedwith samples and screened for ligands that would bind the targetprotein. The method used to screen is described in Hughes et al., U.S.Pat. No. 5,783,397. The screening is conducted by Cetek Corporation,Marlborough, Mass.

EXAMPLE 4 Cloning of S. aureus Uridylate Kinase Coding Sequence forExpression in E. coli

[0100] The S. aureus pyrH coding sequence encoding for uridylate kinasewas cloned into the expression vector pQE60 for production ofrecombinant uridylate kinase in the E. coli strain M15 containing theplasmid pREP4. Cloning of the pyrH coding sequence was by PCR with twooligonucleotide primers 5′CCCGGGCCATGGCTCAAATT (SEQ ID NO: 90) and5′GGGCCCAAGCTTAGTGATGG (SEQ ID NO: 145), using S. aureus genomic DNA asthe template. The PCR product was treated with restriction enzymes NcoIand HindIII, purified by agarose gel electrophoresis, and ligated intopQE60 disgested with NcoI and HindIII. The ligation mixture wastransformed into M15 cells containing pREP4; transformants were selectedand the nucleotide sequence of the pyrH coding sequence was verified byrestriction enzyme analysis and DNA sequencing. The resulting plasmidfor production of S. aureus uridylate kinase in E. coli was designatedpQE60-UMK. Procedures for DNA and plasmid preparation, restrictionenzyme treatment, ligation, and transformation were according to thosedescribed in Sambrook et al. (Molecular Cloning: A Laboratory Manual.,Cold Spring Harbor Laboratory Press (1989)).

[0101] The nucleotide and amino acid sequences of the recombinanturidylate kinase are shown in FIG. 3. Six histidine residues are addedto the C-terminus for purification with Ni-chelating chromatography. Dueto the use of NcoI site for cloning and vector sequence, a alanineresidue is added after the initiation methionine, and arginine andserine residues are added prior to the histidine residues.

EXAMPLE 5 Production of Recombinant S. aureus Uridylate Kinase

[0102] The production strain, designated M15(pQE60-UMK), was grown toA₅₅₀ of about 1 at 30° C. in NS86 medium. NS86 medium is 2.6 grams (g)K₂HPO₄, 10.9 g NaNH₄HPO₄—4H₂O, 2.1 g citric acid, 0.67 g (NH₄)₂SO₄, 0.25g MgSO₄—7H₂O, 10.4 g yeast extract, and 5 g glycerol in 1 liter of H₂O.The NS86 medium was supplemented with 100 μg/ml ampicillin and 25 μg/mlkanamycin. Frozen ampules with 20% glycerol added as a cryoprotectantwere prepared and stored in liquid nitrogen.

[0103] Seed culture was prepared by inoculation of 0.1 ml thawed cellsfrom an ampule into 50 ml of NS86 medium, grown overnight at 30° C., andused to inoculate 100 ml MIM medium. MIM medium is 32 g tryptone, 20 gyeast extract, 6 g Na₂HPO₄, 3 g KH₂PO₄, 0.5 g NaCl, and 1 g NH₄Cl in 1liter of H₂O containing 100 μg/ml ampicillin and 25 μg/ml kanamycin toA₅₅₀ 0.1. Cells were grown at 30° C. overnight to A₅₅₀ 7-8 and used for10-liter fermentation.

[0104] Seed culture was used to inoculate a 10-liter fermentor (NewBrunswick Microgens) with MIM medium to A₅₅₀ 0.1. When cells grew up toa density of A₅₅₀ of about 1 at 30° C., isopropyl-β-D-thiogalactosidewas added to 1 mM to induce the expression of the recombinant protein.Cells were harvested at 2.5 hours post-induction and stored frozen. Theaverage amount of uridylate kinase produced from a 10-liter fermentationwas estimated at 170 mg/l and corresponded to about 20-25% of total cellprotein.

EXAMPLE 6 Purification of Recombinant S. aureus Uridylate Kinase

[0105] The frozen cells from a 10-liter fermentation were thawed andmixed with 200 ml cold lysis buffer (50 mM Tris, pH 7.8 at 22° C., 500mM NaCl, 10% glycerol, 25 mM imidazole, 5 mM 2-mercaptoethanol, 0.1mg/ml DNase). The pellet was homogenized to yield a uniform suspension,and processed two times through a Rainie homogenizer to lyze the cells.The lysed cells were centrifuged at 35,000× g for 75 minutes and thesupernatant liquid was filtered sequentially through Nalgene 0.45 micronand 0.2 micron filters to remove particulates prior to columnchromatography.

[0106] Column chromatography was carried out at 4° C., using a 2.6cm×6.7 cm column packed with Qiagen Ni-NTA Superflow resin. The columnwas washed with 3 bed volumes of water and equilibrated with 3 bedvolumes of equilibration buffer (50 mM Tris, pH 7.8 at 22° C., 500 mMNaCl, 10% glycerol, 5 mM 2-mercaptoethanol) containing 25 mM imidazole.The filtered supernatant was applied to the column at a rate of 3 bedvolumes per hour. After loading, the column was washed withequilibration buffer with 25 mM imidazole until the absorbance at 280nanometers (nm) decreased to 50% of baseline, followed by 6-7 bedvolumes of equilibration buffer plus 40 mM imidazole and then 6-7 bedvolumes of equilibration buffer plus 50 mM imidazole. The bounduridylate kinase was eluted at a rate of 2-3 bed volumes per hour withequilibration buffer plus 300 mM imidazole, and was recovered in fourseparate fractions which were pooled, diluted 3-fold to reduce theprotein concentration, and dialyzed against 50 mM Tris, pH 7.8 at 22°C., 500 mM NaCl, 10% glycerol, 5 mM 2-mercaptoethanol. The dialyzed poolwas stored frozen until further use.

[0107] This isolation yielded about 700 mg of recombinant uridylatekinase protein at a purity of 95-98%. N-terminal sequencing showed thatthe N-terminal methionine was absent, which is expected due to theactivity of host methionine aminopeptidase.

EXAMPLE 7 Enzyme Assays for Uridylate Kinase Activity

[0108] Uridylate kinase catalyzes the transfer of a phosphoryl groupfrom ATP to UMP to form UDP. In the cell, UDP is the substrate/precursorin several metabolic pathways including RNA and synthesis. Aspectrophotometry assay was established by coupling the uridylate kinasereaction to NADH oxidation using pyruvate kinase and lactatedehydrogenase, which ultimately convert the products of uridylate kinasereaction to lactate and NAD⁺. NADH oxidation was monitored by followingthe decrease in absorbance at 340 nm. EDTA at a final concentration of 5mM can be used to stop the assay. This assay was optimized inhigh-throughput format in 96-well microtiter plates to screen for agentsthat inhibit uridylate kinase. A secondary assay for pyruvate kinase andlactate dehydrogenase coupling enzymes was also developed to test thespecificity of agents detected in the primary coupled assay.

[0109] The coupled assay is diagramed below.

[0110] Abbreviations:

[0111] ATP/ADP—Adenosine 5′ triphosphate/Adenosine 5′-diphosphate

[0112] UMP/UDP—Uridine 5′ Monophosphate/Uridine 5′ diphosphate

[0113] PEP—Phospho(enol)pyruvate

[0114] NADH/NAD⁺—Nicotinamide Adenine Dinucleotide Reduced/Oxidized

[0115] The uridylate kinase coupled assay contains the followingreagents in a final volume of 200 μl: assay buffer (50 mM HEPES, pH 7.5,100 mM KCl, 2 mM MgCl₂), 1 mM UMP, 2 mM ATP, 0.22 mM NADH, 2 mM PEP, 3.2units pyruvate kinase (Sigma, St. Louis, Mo.), 4 units lactatedehydrogenase, (Sigma) and 10 ng uridylate kinase. The assay was carriedout at 25° C. with the decrease in absorbance at 340 nm monitored at 15second intervals in the kinetics mode for 1 or 3 hours.

[0116] The complete disclosures of all patents, patent applications,publications, and nucleic acid and protein database entries, includingfor example GenBank accession numbers and EMBL accession numbers, thatare cited herein are hereby incorporated by reference as if individuallyincorporated. Various modifications and alterations of this inventionwill become apparent to those skilled in the art without departing fromthe scope and spirit of this invention, and it should be understood thatthis invention is not to be unduly limited to the illustrativeembodiments set forth herein.

Sequence Listing Free Text

[0117] SEQ ID NO:35-68, 90-108, 111, 112, 115, 116, Oligonucleotideprimer 119, 120, 123, 124, 127, 128, 131, 132, 135, 136, 139, 140,143-145 SEQ ID NO:69, 71, 73, 75, 77, 79, 81, 83, 85 Nucleotide sequenceof S. aureus coding region cloned for expression in E. coli SEQ ID NO:70, 72, 74, 76, 78, 80, 82, Amino acid sequence 84, 86 encoded by S.aureus coding region cloned for expression in E. coli SEQ ID NOs:49, 53,57, 61, 65, 69, 73, Cloned coding 77, 81, 85, 89, 93; sequence SEQ IDNOs:50, 54, 58, 62, 66, 70, 74, Polypeptide 78, 82, 86, 90, 94; encodedby cloned essential coding sequence SEQ ID NO:89 DNA sequence of portionof pQE-60 vector

[0118]

1 145 1 819 DNA Staphylococcus aureus 1 atgtatttac ttacctccaa ttataattgtaccggttcaa tttgtaaacg ccgatacaat 60 tataatattt tgtgctataa taattacagacaaagtgaaa acgaggacag aatattgtta 120 aagtatgaac atattgctaa gcaacttaatgcgtttatac atcaatctaa tttcaaaccc 180 ggtgataaat tgccaagcgt gacgcaattaaaagaacgtt atcaagtaag taagagtact 240 atcattaaag cattaggctt attggaacaagatggtttga tctatcaagc acaaggcagt 300 ggtatttatg tgagaaatat tgctgatgccaatcgtatca acgtctttaa gactaatggt 360 ttctctaaaa gtttaggtga acaccgaatgacaagtaagg tacttgtttt taaggagatt 420 gcaacgccac ctaaatctgt acaagatgagctccaattaa atgcagatga taccgtctac 480 tatttagagc gattaagatt cgtggacgatgatgttttat gtatcgaata ttcttattat 540 cataaagaaa tcgtgaaata tttaaatgatgatattgcta agggctctat cttcgactat 600 ttagaatcaa acatgaaact tcgtattggtttttcagata ttttctttaa tgtagatcaa 660 ctcacttcaa gtgaagcttc attactacaattgtctacag gtgaaccatg tttacgttac 720 caccagactt tttatacaat gactggcaaaccctttgatt catctgacat cgtatttcat 780 tatcgtcatg cacagtttta tattcctagtaaaaagtaa 819 2 272 PRT Staphylococcus aureus 2 Met Tyr Leu Leu Thr SerAsn Tyr Asn Cys Thr Gly Ser Ile Cys Lys 1 5 10 15 Arg Arg Tyr Asn TyrAsn Ile Leu Cys Tyr Asn Asn Tyr Arg Gln Ser 20 25 30 Glu Asn Glu Asp ArgIle Leu Leu Lys Tyr Glu His Ile Ala Lys Gln 35 40 45 Leu Asn Ala Phe IleHis Gln Ser Asn Phe Lys Pro Gly Asp Lys Leu 50 55 60 Pro Ser Val Thr GlnLeu Lys Glu Arg Tyr Gln Val Ser Lys Ser Thr 65 70 75 80 Ile Ile Lys AlaLeu Gly Leu Leu Glu Gln Asp Gly Leu Ile Tyr Gln 85 90 95 Ala Gln Gly SerGly Ile Tyr Val Arg Asn Ile Ala Asp Ala Asn Arg 100 105 110 Ile Asn ValPhe Lys Thr Asn Gly Phe Ser Lys Ser Leu Gly Glu His 115 120 125 Arg MetThr Ser Lys Val Leu Val Phe Lys Glu Ile Ala Thr Pro Pro 130 135 140 LysSer Val Gln Asp Glu Leu Gln Leu Asn Ala Asp Asp Thr Val Tyr 145 150 155160 Tyr Leu Glu Arg Leu Arg Phe Val Asp Asp Asp Val Leu Cys Ile Glu 165170 175 Tyr Ser Tyr Tyr His Lys Glu Ile Val Lys Tyr Leu Asn Asp Asp Ile180 185 190 Ala Lys Gly Ser Ile Phe Asp Tyr Leu Glu Ser Asn Met Lys LeuArg 195 200 205 Ile Gly Phe Ser Asp Ile Phe Phe Asn Val Asp Gln Leu ThrSer Ser 210 215 220 Glu Ala Ser Leu Leu Gln Leu Ser Thr Gly Glu Pro CysLeu Arg Tyr 225 230 235 240 His Gln Thr Phe Tyr Thr Met Thr Gly Lys ProPhe Asp Ser Ser Asp 245 250 255 Ile Val Phe His Tyr Arg His Ala Gln PheTyr Ile Pro Ser Lys Lys 260 265 270 3 828 DNA Staphylococcus aureus 3atggcacttt atggatttgc ccaaggactt attcaagaag caggaattag aattaaacaa 60ttgatggagc aaaatttaac aattgaaaca aagtcaaatc cgaatgacct tgttacaaat 120gtagataaag caacagaaga tttcattttt gatacaattt tagaaacata tcccaatcat 180caagtattag gtgaagaagg gcatggtcat gacatcgata cttccaaagg tacggtatgg 240attgttgacc caatagacgg tacattgaat tttgttcatc aacaagaaaa tttcgcaatt 300tcaattggta tttatatcga tggtaaacct tatgcaggtt ttgtatatga tgttatggct 360gatgtcttat atcatgctaa agtaggggaa ggtgcatatc gtggtagcca acccttgaaa 420ccattgaatg attctaatct aagacaaagc attattggga tcaatccgaa ctggttaact 480aaaccaattt taggagaaat ctttaaagaa attgttaatg attctagaag tgcaagggca 540tatggtagtg cagcgcttga aatcgtttca gttgctacag gtaatttaga agcatatatg 600acgccaagac ttcaaccatg ggattttgct ggcggattgg ttattttata tgaagtaaat 660ggacaagctt ccaatttact aggaggacca ttaacaatta gtggtccaaa ttcaatctta 720gttggaaatc gtggtctcca tcaagaaatt agcaatgatt atttagagcc ccaccatgat 780gcgttaatac aattacatga acaacgattt aaaagaaaat caaaataa 828 4 275 PRTStaphylococcus aureus 4 Met Ala Leu Tyr Gly Phe Ala Gln Gly Leu Ile GlnGlu Ala Gly Ile 1 5 10 15 Arg Ile Lys Gln Leu Met Glu Gln Asn Leu ThrIle Glu Thr Lys Ser 20 25 30 Asn Pro Asn Asp Leu Val Thr Asn Val Asp LysAla Thr Glu Asp Phe 35 40 45 Ile Phe Asp Thr Ile Leu Glu Thr Tyr Pro AsnHis Gln Val Leu Gly 50 55 60 Glu Glu Gly His Gly His Asp Ile Asp Thr SerLys Gly Thr Val Trp 65 70 75 80 Ile Val Asp Pro Ile Asp Gly Thr Leu AsnPhe Val His Gln Gln Glu 85 90 95 Asn Phe Ala Ile Ser Ile Gly Ile Tyr IleAsp Gly Lys Pro Tyr Ala 100 105 110 Gly Phe Val Tyr Asp Val Met Ala AspVal Leu Tyr His Ala Lys Val 115 120 125 Gly Glu Gly Ala Tyr Arg Gly SerGln Pro Leu Lys Pro Leu Asn Asp 130 135 140 Ser Asn Leu Arg Gln Ser IleIle Gly Ile Asn Pro Asn Trp Leu Thr 145 150 155 160 Lys Pro Ile Leu GlyGlu Ile Phe Lys Glu Ile Val Asn Asp Ser Arg 165 170 175 Ser Ala Arg AlaTyr Gly Ser Ala Ala Leu Glu Ile Val Ser Val Ala 180 185 190 Thr Gly AsnLeu Glu Ala Tyr Met Thr Pro Arg Leu Gln Pro Trp Asp 195 200 205 Phe AlaGly Gly Leu Val Ile Leu Tyr Glu Val Asn Gly Gln Ala Ser 210 215 220 AsnLeu Leu Gly Gly Pro Leu Thr Ile Ser Gly Pro Asn Ser Ile Leu 225 230 235240 Val Gly Asn Arg Gly Leu His Gln Glu Ile Ser Asn Asp Tyr Leu Glu 245250 255 Pro His His Asp Ala Leu Ile Gln Leu His Glu Gln Arg Phe Lys Arg260 265 270 Lys Ser Lys 275 5 543 DNA Staphylococcus aureus 5 atgggattcaaaaacaattt aacatcaaat ttaacaaata aaatcggtaa ttcagtcttt 60 aaaatagaaaatgttgacgg aaaaggtgca atgccaacga cgattcaaga attgagagaa 120 agacgacaacgtgctgaagc aattgtaaag agaaagtctt taatgtcatc aacaatgagc 180 gttgttccaattccgggttt agattttggt gttgatttaa aattaatgaa agatattatc 240 gaagatgttaataaaattta tggtttagat cataagcaag ttaatagcct tggggatgat 300 gtgaaagaaagaattatgtc tgcagcagca attcaaggta gtcaatttat tggtaaaaga 360 atttcaaatgcatttttaaa aattgtaatt agagatgtag ctaaacgtac tgctgcaaaa 420 caaacaaaatggtttcctgt tgtaggacaa gctgtgtctg catctattag ttactatttt 480 atgaataaaattggaaaaga tcacattcaa aaatgcgaaa atgttattaa aaatgtcatg 540 tag 543 6 180PRT Staphylococcus aureus 6 Met Gly Phe Lys Asn Asn Leu Thr Ser Asn LeuThr Asn Lys Ile Gly 1 5 10 15 Asn Ser Val Phe Lys Ile Glu Asn Val AspGly Lys Gly Ala Met Pro 20 25 30 Thr Thr Ile Gln Glu Leu Arg Glu Arg ArgGln Arg Ala Glu Ala Ile 35 40 45 Val Lys Arg Lys Ser Leu Met Ser Ser ThrMet Ser Val Val Pro Ile 50 55 60 Pro Gly Leu Asp Phe Gly Val Asp Leu LysLeu Met Lys Asp Ile Ile 65 70 75 80 Glu Asp Val Asn Lys Ile Tyr Gly LeuAsp His Lys Gln Val Asn Ser 85 90 95 Leu Gly Asp Asp Val Lys Glu Arg IleMet Ser Ala Ala Ala Ile Gln 100 105 110 Gly Ser Gln Phe Ile Gly Lys ArgIle Ser Asn Ala Phe Leu Lys Ile 115 120 125 Val Ile Arg Asp Val Ala LysArg Thr Ala Ala Lys Gln Thr Lys Trp 130 135 140 Phe Pro Val Val Gly GlnAla Val Ser Ala Ser Ile Ser Tyr Tyr Phe 145 150 155 160 Met Asn Lys IleGly Lys Asp His Ile Gln Lys Cys Glu Asn Val Ile 165 170 175 Lys Asn ValMet 180 7 936 DNA Staphylococcus aureus 7 gtgtttcatc atatcagcgttatgttaaac gaaaccattg attatttaaa tgtaaaagaa 60 aatggtgtgt acattgactgtacgctaggt ggagcgggac atgcccttta tttactaaat 120 caattaaatg acgacggaagattaatagca atcgatcaag accaaactgc aattgataat 180 gctaaagagg tattaaaggatcatttgcat aaggtgactt ttgttcatag caacttccgt 240 gaattaactc aaatattaaaagacttaaac attgaaaaag tagatggaat ttattacgac 300 ttgggtgttt caagcccacaactcgacatt ccagaacgag gattcagtta tcaccatgac 360 gcaacattag acatgcgtatggaccaaaca caagaactaa cagcatatga aattgttaac 420 aattggtcat atgaagcgttagtgaagatt ttttatcgct atggcgagga gaaattttca 480 aaacagatag ctcgaagaatcgaagcacat cgcgaacaac aaccaataac aacaacatta 540 gaattagttg acattataaaagaaggtatt cctgcaaaag caagaagaaa aggcggacat 600 cctgcaaaac gagtatttcaagcactacga attgcagtaa acgatgaatt gtcagctttt 660 gaagattcaa tagaacaagcgattgaatta gtgaaagtag atggcaggat ttcggtaatc 720 actttccatt ctttagaagatcgtttatgt aaacaggtgt tccaagaata tgaaaaaggt 780 ccagaggtac caagaggattaccagttata ccagaagcat atacacctaa gttaaagcgt 840 gttaatcgta aaccgattaccgctacagaa gaagatttag atgacaataa cagagcacga 900 agcgcgaaat tacgtgtagctgaaatactt aaataa 936 8 311 PRT Staphylococcus aureus 8 Val Phe His HisIle Ser Val Met Leu Asn Glu Thr Ile Asp Tyr Leu 1 5 10 15 Asn Val LysGlu Asn Gly Val Tyr Ile Asp Cys Thr Leu Gly Gly Ala 20 25 30 Gly His AlaLeu Tyr Leu Leu Asn Gln Leu Asn Asp Asp Gly Arg Leu 35 40 45 Ile Ala IleAsp Gln Asp Gln Thr Ala Ile Asp Asn Ala Lys Glu Val 50 55 60 Leu Lys AspHis Leu His Lys Val Thr Phe Val His Ser Asn Phe Arg 65 70 75 80 Glu LeuThr Gln Ile Leu Lys Asp Leu Asn Ile Glu Lys Val Asp Gly 85 90 95 Ile TyrTyr Asp Leu Gly Val Ser Ser Pro Gln Leu Asp Ile Pro Glu 100 105 110 ArgGly Phe Ser Tyr His His Asp Ala Thr Leu Asp Met Arg Met Asp 115 120 125Gln Thr Gln Glu Leu Thr Ala Tyr Glu Ile Val Asn Asn Trp Ser Tyr 130 135140 Glu Ala Leu Val Lys Ile Phe Tyr Arg Tyr Gly Glu Glu Lys Phe Ser 145150 155 160 Lys Gln Ile Ala Arg Arg Ile Glu Ala His Arg Glu Gln Gln ProIle 165 170 175 Thr Thr Thr Leu Glu Leu Val Asp Ile Ile Lys Glu Gly IlePro Ala 180 185 190 Lys Ala Arg Arg Lys Gly Gly His Pro Ala Lys Arg ValPhe Gln Ala 195 200 205 Leu Arg Ile Ala Val Asn Asp Glu Leu Ser Ala PheGlu Asp Ser Ile 210 215 220 Glu Gln Ala Ile Glu Leu Val Lys Val Asp GlyArg Ile Ser Val Ile 225 230 235 240 Thr Phe His Ser Leu Glu Asp Arg LeuCys Lys Gln Val Phe Gln Glu 245 250 255 Tyr Glu Lys Gly Pro Glu Val ProArg Gly Leu Pro Val Ile Pro Glu 260 265 270 Ala Tyr Thr Pro Lys Leu LysArg Val Asn Arg Lys Pro Ile Thr Ala 275 280 285 Thr Glu Glu Asp Leu AspAsp Asn Asn Arg Ala Arg Ser Ala Lys Leu 290 295 300 Arg Val Ala Glu IleLeu Lys 305 310 9 969 DNA Staphylococcus aureus 9 atgataaata atcatgaattactaggtatt caccatgtta ctgcaatgac agatgatgca 60 gaacgtaatt ataaattttttacagaagta ctaggcatgc gtttagttaa aaagacagtc 120 aatcaagatg atatttatacgtatcatact ttttttgcag atgatgtagg ttcggcaggt 180 acagacatga cgttctttgattttccaaat attacaaaag ggcaggcagg aacaaattcc 240 attacaagac cgtcttttagagtgcctaac gatgacgcat taacatatta tgaacagcgc 300 tttgatgagt ttggtgttaaacacgaaggt attcaagaat tatttggtaa aaaagtgttg 360 ccatttgaag aagtcgatggccaagtgtat caattaattt cagatgagtt aaatgaaggg 420 gtagcacctg gtgtaccttggaagaatgga ccggttccag tagataaagc gatttatgga 480 ttaggcccca ttgaaattaaagtaagttat tttgacgact ttaaaaatat tttagagact 540 gtttacggta tgacaactattgcgcatgaa gataatgtcg cattacttga agttggcgaa 600 ggaggcaatg gtggccaggtaatcttaata aaagatgata aagggccagc agcacgtcaa 660 ggttatggtg aggtacatcatgtgtcattt cgtgtgaaag atcatgatgc aatagaagcg 720 tgggcaacga aatataaagaggtaggtatt aataactcag gcatcgttaa tcgtttctat 780 tttgaagcat tatatgcacgtgtggggcat attttaatag aaatttcaac agatggacca 840 ggatttatgg aagatgaaccttatgaaaca ttaggcgaag ggttatcctt accaccattt 900 ttagaaaata aaagagaatatattgaatcg gaagttagac cttttaatac gaagcgtcaa 960 catggttaa 969 10 322 PRTStaphylococcus aureus 10 Met Ile Asn Asn His Glu Leu Leu Gly Ile His HisVal Thr Ala Met 1 5 10 15 Thr Asp Asp Ala Glu Arg Asn Tyr Lys Phe PheThr Glu Val Leu Gly 20 25 30 Met Arg Leu Val Lys Lys Thr Val Asn Gln AspAsp Ile Tyr Thr Tyr 35 40 45 His Thr Phe Phe Ala Asp Asp Val Gly Ser AlaGly Thr Asp Met Thr 50 55 60 Phe Phe Asp Phe Pro Asn Ile Thr Lys Gly GlnAla Gly Thr Asn Ser 65 70 75 80 Ile Thr Arg Pro Ser Phe Arg Val Pro AsnAsp Asp Ala Leu Thr Tyr 85 90 95 Tyr Glu Gln Arg Phe Asp Glu Phe Gly ValLys His Glu Gly Ile Gln 100 105 110 Glu Leu Phe Gly Lys Lys Val Leu ProPhe Glu Glu Val Asp Gly Gln 115 120 125 Val Tyr Gln Leu Ile Ser Asp GluLeu Asn Glu Gly Val Ala Pro Gly 130 135 140 Val Pro Trp Lys Asn Gly ProVal Pro Val Asp Lys Ala Ile Tyr Gly 145 150 155 160 Leu Gly Pro Ile GluIle Lys Val Ser Tyr Phe Asp Asp Phe Lys Asn 165 170 175 Ile Leu Glu ThrVal Tyr Gly Met Thr Thr Ile Ala His Glu Asp Asn 180 185 190 Val Ala LeuLeu Glu Val Gly Glu Gly Gly Asn Gly Gly Gln Val Ile 195 200 205 Leu IleLys Asp Asp Lys Gly Pro Ala Ala Arg Gln Gly Tyr Gly Glu 210 215 220 ValHis His Val Ser Phe Arg Val Lys Asp His Asp Ala Ile Glu Ala 225 230 235240 Trp Ala Thr Lys Tyr Lys Glu Val Gly Ile Asn Asn Ser Gly Ile Val 245250 255 Asn Arg Phe Tyr Phe Glu Ala Leu Tyr Ala Arg Val Gly His Ile Leu260 265 270 Ile Glu Ile Ser Thr Asp Gly Pro Gly Phe Met Glu Asp Glu ProTyr 275 280 285 Glu Thr Leu Gly Glu Gly Leu Ser Leu Pro Pro Phe Leu GluAsn Lys 290 295 300 Arg Glu Tyr Ile Glu Ser Glu Val Arg Pro Phe Asn ThrLys Arg Gln 305 310 315 320 His Gly 11 1100 DNA Staphylococcus aureus 11gggacatttt taaatcatgc atgcgtatct taaaagagtc cattattgtg gcatttgcct 60ttgttggtgt tgtcgttggt gccggctttg ctactggtca agaaattttc cagtttttca 120caagtcatgg cgcatatagc atttcaggca ttattgtaac aggactattg attactttag 180gtggaatggt tgtcatgcat acaggtcatc atctaaagtc cagaaatcat tctgattcaa 240ttaactattt cttatacccc tctattgcaa gaggttttga tattatttta acaatgttta 300tgttgtcttt agctattatt atgactgcag gtggtgcgtc aaccattcat caaagtttca 360acttaccgta ttggctgagc gcactcatat tagtcgcctt tattttagca acactgtttc 420taaaattcga tcgtttaatt gctgtgcttg gcggtgttac cccattttta attgcgattg 480tcattatgat tgcggtctac tatttcacaa caagtcatct tgattttact gccgctaata 540atgatgctca gattcataag cagaaatcat tatcacctgg atggtggttt gatgcgatta 600actatgcaag cttgcaaatt gctgctgcct tcagcttctt atcagtgatg ggtagtaaag 660ttaaatatcg tgactcaacg ttatacgggg gcttgattgg cggtttaatc attacatttt 720tactcatgat gattaatcta ggtttaattt ctcaattcga taaaattaaa cacgtagatc 780tacctacatt aaaattagcg acacaaatgt ctccgtcaat tggtattatt atgtctgtca 840ttatgatact tgtcatctac aatactgttg ttggattaat gtatgcattt gcgtcacgtt 900tcagcgttcc attcagcaga cgttacttca tcattattat tacaatggct gtcatcactt 960atattagtac atttatcggt ttcatttcat taattggaaa agtattccct attatgggat 1020tgttcggttt catcttactc atacctgtac tctataaagg tttaattaag cgtattaccg 1080gcaaatctca tatcgattaa 1100 12 359 PRT Staphylococcus aureus 12 Met ArgIle Leu Lys Glu Ser Ile Ile Val Ala Phe Ala Phe Val Gly 1 5 10 15 ValVal Val Gly Ala Gly Phe Ala Thr Gly Gln Glu Ile Phe Gln Phe 20 25 30 PheThr Ser His Gly Ala Tyr Ser Ile Ser Gly Ile Ile Val Thr Gly 35 40 45 LeuLeu Ile Thr Leu Gly Gly Met Val Val Met His Thr Gly His His 50 55 60 LeuLys Ser Arg Asn His Ser Asp Ser Ile Asn Tyr Phe Leu Tyr Pro 65 70 75 80Ser Ile Ala Arg Gly Phe Asp Ile Ile Leu Thr Met Phe Met Leu Ser 85 90 95Leu Ala Ile Ile Met Thr Ala Gly Gly Ala Ser Thr Ile His Gln Ser 100 105110 Phe Asn Leu Pro Tyr Trp Leu Ser Ala Leu Ile Leu Val Ala Phe Ile 115120 125 Leu Ala Thr Leu Phe Leu Lys Phe Asp Arg Leu Ile Ala Val Leu Gly130 135 140 Gly Val Thr Pro Phe Leu Ile Ala Ile Val Ile Met Ile Ala ValTyr 145 150 155 160 Tyr Phe Thr Thr Ser His Leu Asp Phe Thr Ala Ala AsnAsn Asp Ala 165 170 175 Gln Ile His Lys Gln Lys Ser Leu Ser Pro Gly TrpTrp Phe Asp Ala 180 185 190 Ile Asn Tyr Ala Ser Leu Gln Ile Ala Ala AlaPhe Ser Phe Leu Ser 195 200 205 Val Met Gly Ser Lys Val Lys Tyr Arg AspSer Thr Leu Tyr Gly Gly 210 215 220 Leu Ile Gly Gly Leu Ile Ile Thr PheLeu Leu Met Met Ile Asn Leu 225 230 235 240 Gly Leu Ile Ser Gln Phe AspLys Ile Lys His Val Asp Leu Pro Thr 245 250 255 Leu Lys Leu Ala Thr GlnMet Ser Pro Ser Ile Gly Ile Ile Met Ser 260 265 270 Val Ile Met Ile LeuVal Ile Tyr Asn Thr Val Val Gly Leu Met Tyr 275 280 285 Ala Phe Ala SerArg Phe Ser Val Pro Phe Ser Arg Arg Tyr Phe Ile 290 295 300 Ile Ile IleThr Met Ala Val Ile Thr Tyr Ile Ser Thr Phe Ile Gly 305 310 315 320 PheIle Ser Leu Ile Gly Lys Val Phe Pro Ile Met Gly Leu Phe Gly 325 330 335Phe Ile Leu Leu Ile Pro Val Leu Tyr Lys Gly Leu Ile Lys Arg Ile 340 345350 Thr Gly Lys Ser His Ile Asp 355 13 774 DNA Staphylococcus aureus 13atgttaatcg atacacatgt ccatttaaat gatgagcaat acgatgatga tttgagtgaa 60gtgattacac gtgctagaga agcaggtgtt gatcgtatgt ttgtagttgg ttttaacaaa 120tcgacaattg aacgcgcgat gaaattaatc gatgagtatg attttttata tggcattatc 180ggttggcatc cagttgacgc aattgatttt acagaagaac acttggaatg gattgaatct 240ttagctcagc atccaaaagt gattggtatt ggtgaaatgg gattagatta tcactgggat 300aaatctcctg cagatgttca aaaggaagtt tttagaaagc aaattgcttt agctaagcgt 360ttgaagttac caattatcat tcataaccgt gaagcaactc aagactgtat cgatatctta 420ttggaggagc atgctgaaga ggtaggcggg attatgcata gctttagtgg ttctccagaa 480attgcagata ttgtaactaa taagctgaat ttttatattt cattaggtgg acctgtgaca 540tttaaaaatg ctaaacagcc taaagaagtt gctaagcatg tgtcaatgga gcgtttgcta 600gttgaaaccg atgcaccgta tctttcgcca catccgtata gagggaagcg aaatgaaccg 660gcgagagtaa ctttagtagc tgaacaaatt gctgaattaa aaggcttatc ttatgaagaa 720gtgtgcgaac aaacaactaa aaatgcagag aaattgttta atttaaattc ataa 774 14 257PRT Staphylococcus aureus 14 Met Leu Ile Asp Thr His Val His Leu Asn AspGlu Gln Tyr Asp Asp 1 5 10 15 Asp Leu Ser Glu Val Ile Thr Arg Ala ArgGlu Ala Gly Val Asp Arg 20 25 30 Met Phe Val Val Gly Phe Asn Lys Ser ThrIle Glu Arg Ala Met Lys 35 40 45 Leu Ile Asp Glu Tyr Asp Phe Leu Tyr GlyIle Ile Gly Trp His Pro 50 55 60 Val Asp Ala Ile Asp Phe Thr Glu Glu HisLeu Glu Trp Ile Glu Ser 65 70 75 80 Leu Ala Gln His Pro Lys Val Ile GlyIle Gly Glu Met Gly Leu Asp 85 90 95 Tyr His Trp Asp Lys Ser Pro Ala AspVal Gln Lys Glu Val Phe Arg 100 105 110 Lys Gln Ile Ala Leu Ala Lys ArgLeu Lys Leu Pro Ile Ile Ile His 115 120 125 Asn Arg Glu Ala Thr Gln AspCys Ile Asp Ile Leu Leu Glu Glu His 130 135 140 Ala Glu Glu Val Gly GlyIle Met His Ser Phe Ser Gly Ser Pro Glu 145 150 155 160 Ile Ala Asp IleVal Thr Asn Lys Leu Asn Phe Tyr Ile Ser Leu Gly 165 170 175 Gly Pro ValThr Phe Lys Asn Ala Lys Gln Pro Lys Glu Val Ala Lys 180 185 190 His ValSer Met Glu Arg Leu Leu Val Glu Thr Asp Ala Pro Tyr Leu 195 200 205 SerPro His Pro Tyr Arg Gly Lys Arg Asn Glu Pro Ala Arg Val Thr 210 215 220Leu Val Ala Glu Gln Ile Ala Glu Leu Lys Gly Leu Ser Tyr Glu Glu 225 230235 240 Val Cys Glu Gln Thr Thr Lys Asn Ala Glu Lys Leu Phe Asn Leu Asn245 250 255 Ser 15 2123 DNA Staphylococcus aureus 15 atgataatatattggtgtat gacagttaat ggagggaacg aaatgaaagc tttattactt 60 aaaacaagtgtatggctcgt tttgcttttt agtgtaatgg gattatggca agtctcgaac 120 gcggctgagcagcatacacc aatgaaagca catgcagtaa caacgataga caaagcaaca 180 acagataagcaacaagtacc gccaacaaag gaagcggctc atcattctgg caaagaagcg 240 gcaaccaacgtatcagcatc agcgcaggga acagctgatg atacaaacag caaagtaaca 300 tccaacgcaccatctaacaa accatctaca gtagtttcaa caaaagtaaa cgaaacacgc 360 gacgtagatacacaacaagc ctcaacacaa aaaccaactc acacagcaac gttcaaatta 420 tcaaatgctaaaacagcatc actttcacca cgaatgtttg ctgctaatgc accacaaaca 480 acaacacataaaatattaca tacaaatgat atccatggcc gactagccga agaaaaaggg 540 cgtgtcatcggtatggctaa attaaaaaca gtaaaagaac aagaaaagcc tgatttaatg 600 ttagacgcaggagacgcctt ccaaggttta ccactttcaa accagtctaa aggtgaagaa 660 atggctaaagcaatgaatgc agtaggttat gatgctatgg cagtcggtaa ccatgaattt 720 gactttggatacgatcagtt gaaaaagtta gagggtatgt tagacttccc gatgctaagt 780 actaacgtttataaagatgg aaaacgcgcg tttaagcctt caacgattgt aacaaaaaat 840 ggtattcgttatggaattat tggtgtaacg acaccagaaa caaagacgaa aacaagacct 900 gaaggcattaaaggcgttga atttagagat ccattacaaa gtgtgacagc ggaaatgatg 960 cgtatttataaagacgtaga tacatttgtt gttatatcac atttaggaat tgatccttca 1020 acacaagaaacatggcgtgg tgattactta gtgaaacaat taagtcaaaa tccacaattg 1080 aagaaacgtattacagttat tgatggtcat tcacatacag tacttcaaaa tggtcaaatt 1140 tataacaatgatgcattggc acaaacaggt acagcacttg cgaatatcgg taagattaca 1200 tttaattatcgcaatggaga ggtatcgaat attaaaccgt cattgattaa tgttaaagac 1260 gttgaaaatgtaacaccgaa caaagcatta gctgaacaaa ttaatcaagc tgatcaaaca 1320 tttagagcacaaactgcaga ggtaattatt ccaaacaata ccattgattt caaaggagaa 1380 agagatgacgttagaacgcg tgaaacaaat ttaggaaacg cgattgcaga tgctatggaa 1440 gcgtatggcgttaagaattt ctctaaaaag actgactttg ccgtgacaaa tggtggaggt 1500 attcgtgcctctatcgcaaa aggtaaggtg acacgctatg atttaatctc agtattacca 1560 tttggaaatacgattgcgca aattgatgta aaaggttcag acgtctggac ggctttcgaa 1620 catagtttaggcgcaccaac aacacaaaag gacggtaaga cagtgttaac agcgaatggc 1680 ggtttactacatatctctga ttcaatccgt gtttactatg atataaataa accgtctggc 1740 aaacgaattaatgctattca aattttaaat aaagagacag gtaagtttga aaatattgat 1800 ttaaaacgtgtatatcacgt aacgatgaat gacttcacag catcaggtgg gacggatata 1860 gtatgttcggtggtcctaga gaagaaggta tttcattaga tcaagtacta gcaagttatt 1920 taaaaacagctaacttagct aagtatgata cgacagaacc acaacgtatg ttattaggta 1980 aaccagcagtaagtgaacaa ccagctaaag gacaacaagg tagcaaaggt agtaagtctg 2040 gtaaagatacacaaccaatt ggtgacgaca aagtgatgga tccagcgaaa aaaccagctc 2100 caggtaaagttgtattgttg tag 2123 16 707 PRT Staphylococcus aureus 16 Met Ile Ile TyrTrp Cys Met Thr Val Asn Gly Gly Asn Glu Met Lys 1 5 10 15 Ala Leu LeuLeu Lys Thr Ser Val Trp Leu Val Leu Leu Phe Ser Val 20 25 30 Met Gly LeuTrp Gln Val Ser Asn Ala Ala Glu Gln His Thr Pro Met 35 40 45 Lys Ala HisAla Val Thr Thr Ile Asp Lys Ala Thr Thr Asp Lys Gln 50 55 60 Gln Val ProPro Thr Lys Glu Ala Ala His His Ser Gly Lys Glu Ala 65 70 75 80 Ala ThrAsn Val Ser Ala Ser Ala Gln Gly Thr Ala Asp Asp Thr Asn 85 90 95 Ser LysVal Thr Ser Asn Ala Pro Ser Asn Lys Pro Ser Thr Val Val 100 105 110 SerThr Lys Val Asn Glu Thr Arg Asp Val Asp Thr Gln Gln Ala Ser 115 120 125Thr Gln Lys Pro Thr His Thr Ala Thr Phe Lys Leu Ser Asn Ala Lys 130 135140 Thr Ala Ser Leu Ser Pro Arg Met Phe Ala Ala Asn Ala Pro Gln Thr 145150 155 160 Thr Thr His Lys Ile Leu His Thr Asn Asp Ile His Gly Arg LeuAla 165 170 175 Glu Glu Lys Gly Arg Val Ile Gly Met Ala Lys Leu Lys ThrVal Lys 180 185 190 Glu Gln Glu Lys Pro Asp Leu Met Leu Asp Ala Gly AspAla Phe Gln 195 200 205 Gly Leu Pro Leu Ser Asn Gln Ser Lys Gly Glu GluMet Ala Lys Ala 210 215 220 Met Asn Ala Val Gly Tyr Asp Ala Met Ala ValGly Asn His Glu Phe 225 230 235 240 Asp Phe Gly Tyr Asp Gln Leu Lys LysLeu Glu Gly Met Leu Asp Phe 245 250 255 Pro Met Leu Ser Thr Asn Val TyrLys Asp Gly Lys Arg Ala Phe Lys 260 265 270 Pro Ser Thr Ile Val Thr LysAsn Gly Ile Arg Tyr Gly Ile Ile Gly 275 280 285 Val Thr Thr Pro Glu ThrLys Thr Lys Thr Arg Pro Glu Gly Ile Lys 290 295 300 Gly Val Glu Phe ArgAsp Pro Leu Gln Ser Val Thr Ala Glu Met Met 305 310 315 320 Arg Ile TyrLys Asp Val Asp Thr Phe Val Val Ile Ser His Leu Gly 325 330 335 Ile AspPro Ser Thr Gln Glu Thr Trp Arg Gly Asp Tyr Leu Val Lys 340 345 350 GlnLeu Ser Gln Asn Pro Gln Leu Lys Lys Arg Ile Thr Val Ile Asp 355 360 365Gly His Ser His Thr Val Leu Gln Asn Gly Gln Ile Tyr Asn Asn Asp 370 375380 Ala Leu Ala Gln Thr Gly Thr Ala Leu Ala Asn Ile Gly Lys Ile Thr 385390 395 400 Phe Asn Tyr Arg Asn Gly Glu Val Ser Asn Ile Lys Pro Ser LeuIle 405 410 415 Asn Val Lys Asp Val Glu Asn Val Thr Pro Asn Lys Ala LeuAla Glu 420 425 430 Gln Ile Asn Gln Ala Asp Gln Thr Phe Arg Ala Gln ThrAla Glu Val 435 440 445 Ile Ile Pro Asn Asn Thr Ile Asp Phe Lys Gly GluArg Asp Asp Val 450 455 460 Arg Thr Arg Glu Thr Asn Leu Gly Asn Ala IleAla Asp Ala Met Glu 465 470 475 480 Ala Tyr Gly Val Lys Asn Phe Ser LysLys Thr Asp Phe Ala Val Thr 485 490 495 Asn Gly Gly Gly Ile Arg Ala SerIle Ala Lys Gly Lys Val Thr Arg 500 505 510 Tyr Asp Leu Ile Ser Val LeuPro Phe Gly Asn Thr Ile Ala Gln Ile 515 520 525 Asp Val Lys Gly Ser AspVal Trp Thr Ala Phe Glu His Ser Leu Gly 530 535 540 Ala Pro Thr Thr GlnLys Asp Gly Lys Thr Val Leu Thr Ala Asn Gly 545 550 555 560 Gly Leu LeuHis Ile Ser Asp Ser Ile Arg Val Tyr Tyr Asp Ile Asn 565 570 575 Lys ProSer Gly Lys Arg Ile Asn Ala Ile Gln Ile Leu Asn Lys Glu 580 585 590 ThrGly Lys Phe Glu Asn Ile Asp Leu Lys Arg Val Tyr His Val Thr 595 600 605Met Asn Asp Phe Thr Ala Ser Gly Gly Asp Gly Tyr Ser Met Phe Gly 610 615620 Gly Pro Arg Glu Glu Gly Ile Ser Leu Asp Gln Val Leu Ala Ser Tyr 625630 635 640 Leu Lys Thr Ala Asn Leu Ala Lys Tyr Asp Thr Thr Glu Pro GlnArg 645 650 655 Met Leu Leu Gly Lys Pro Ala Val Ser Glu Gln Pro Ala LysGly Gln 660 665 670 Gln Gly Ser Lys Gly Ser Lys Ser Gly Lys Asp Thr GlnPro Ile Gly 675 680 685 Asp Asp Lys Val Met Asp Pro Ala Lys Lys Pro AlaPro Gly Lys Val 690 695 700 Val Leu Leu 705 17 1482 DNA Staphylococcusaureus 17 atgcgattta cattttcaaa cgatttagga acgttattta ctattattttagccattgga 60 ttcatcatta atttagtatt ggcttttatt attatctttt tagaaagaaataggcgtaca 120 gcgagttcaa cttgggcatg gctatttgta ctttttgtct taccattgattggttttatt 180 ctttacttgt tttttggtag aaccgtttcg gcacgcaaat tgaataaaaacaatggtaac 240 gtgttaacgg atttcgatgg acttttaaaa caacaaatag aaagctttgataaaggtaat 300 tatggtactg ataacaaaca agttcaaaaa catcatgatt tagtacgtatgcttttgatg 360 gatcaagatg gttttttaac tgaaaataat aaagttgatc atttcattgatggaaatgat 420 ttatatgatc aagttttaaa agatattaaa aatgcaaaag aatatatccatttagagtac 480 tatactttcg ctttagatgg tttaggtaaa agaattttac atgctttagaagaaaaattg 540 aaacaaggtc tagaagtaaa aatattatat gatgatgttg gatctaaaaatgttaagatg 600 gcaaattttg atcattttaa atcgttaggt ggagaagttg aagcattttttgcttcaaaa 660 ttaccgttat tgaatttcag aatgaataat agaaatcata gaaaaatcatcgtaatcgat 720 ggtcaactag gttatgtcgg aggatttaac attggtgatg aatatctaggattaggaaaa 780 ttaggatatt ggagagatac gcatttacgt atacaagggg atgcggttgatgcactgcag 840 ttgcgattta ttttagactg gaattcgcaa gcgcaccgtc cacaatttgaatatgatgtt 900 aagtatttcc ctaaaaagaa cggaccattg ggcaattcac caattcaaatagctgcaagt 960 ggcccggcta gtgactggca tcaaattgaa tacggttata caaaaatgattatgagtgca 1020 aagaaatctg tatatttaca atcaccatat ttcattccgg ataattcatatataaatgcc 1080 attaaaattg ctgctaaatc aggtgtagat gtacatttaa tgattccatgtaagccagat 1140 catccattag tatattgggc gacattttca aatgcctctg acttattatcaagtggtgtt 1200 aaaatttata cgtatgaaaa tggatttata cattctaaaa tgtgcttaattgatgatgaa 1260 atcgtatcag tgggcacagc aaatatggac tttagaagtt ttgaattaaattttgaagta 1320 aatgcctttg tatatgatga aaatcttgct aaagatttaa gggtggcttatgaacatgat 1380 attacaaaat caaaacaact aaccaaagaa tcatatgcca atagaccgctgtctgttaaa 1440 ttcaaagaat cgttagcaaa attagtttcg ccaattttat aa 1482 18493 PRT Staphylococcus aureus 18 Met Arg Phe Thr Phe Ser Asn Asp Leu GlyThr Leu Phe Thr Ile Ile 1 5 10 15 Leu Ala Ile Gly Phe Ile Ile Asn LeuVal Leu Ala Phe Ile Ile Ile 20 25 30 Phe Leu Glu Arg Asn Arg Arg Thr AlaSer Ser Thr Trp Ala Trp Leu 35 40 45 Phe Val Leu Phe Val Leu Pro Leu IleGly Phe Ile Leu Tyr Leu Phe 50 55 60 Phe Gly Arg Thr Val Ser Ala Arg LysLeu Asn Lys Asn Asn Gly Asn 65 70 75 80 Val Leu Thr Asp Phe Asp Gly LeuLeu Lys Gln Gln Ile Glu Ser Phe 85 90 95 Asp Lys Gly Asn Tyr Gly Thr AspAsn Lys Gln Val Gln Lys His His 100 105 110 Asp Leu Val Arg Met Leu LeuMet Asp Gln Asp Gly Phe Leu Thr Glu 115 120 125 Asn Asn Lys Val Asp HisPhe Ile Asp Gly Asn Asp Leu Tyr Asp Gln 130 135 140 Val Leu Lys Asp IleLys Asn Ala Lys Glu Tyr Ile His Leu Glu Tyr 145 150 155 160 Tyr Thr PheAla Leu Asp Gly Leu Gly Lys Arg Ile Leu His Ala Leu 165 170 175 Glu GluLys Leu Lys Gln Gly Leu Glu Val Lys Ile Leu Tyr Asp Asp 180 185 190 ValGly Ser Lys Asn Val Lys Met Ala Asn Phe Asp His Phe Lys Ser 195 200 205Leu Gly Gly Glu Val Glu Ala Phe Phe Ala Ser Lys Leu Pro Leu Leu 210 215220 Asn Phe Arg Met Asn Asn Arg Asn His Arg Lys Ile Ile Val Ile Asp 225230 235 240 Gly Gln Leu Gly Tyr Val Gly Gly Phe Asn Ile Gly Asp Glu TyrLeu 245 250 255 Gly Leu Gly Lys Leu Gly Tyr Trp Arg Asp Thr His Leu ArgIle Gln 260 265 270 Gly Asp Ala Val Asp Ala Leu Gln Leu Arg Phe Ile LeuAsp Trp Asn 275 280 285 Ser Gln Ala His Arg Pro Gln Phe Glu Tyr Asp ValLys Tyr Phe Pro 290 295 300 Lys Lys Asn Gly Pro Leu Gly Asn Ser Pro IleGln Ile Ala Ala Ser 305 310 315 320 Gly Pro Ala Ser Asp Trp His Gln IleGlu Tyr Gly Tyr Thr Lys Met 325 330 335 Ile Met Ser Ala Lys Lys Ser ValTyr Leu Gln Ser Pro Tyr Phe Ile 340 345 350 Pro Asp Asn Ser Tyr Ile AsnAla Ile Lys Ile Ala Ala Lys Ser Gly 355 360 365 Val Asp Val His Leu MetIle Pro Cys Lys Pro Asp His Pro Leu Val 370 375 380 Tyr Trp Ala Thr PheSer Asn Ala Ser Asp Leu Leu Ser Ser Gly Val 385 390 395 400 Lys Ile TyrThr Tyr Glu Asn Gly Phe Ile His Ser Lys Met Cys Leu 405 410 415 Ile AspAsp Glu Ile Val Ser Val Gly Thr Ala Asn Met Asp Phe Arg 420 425 430 SerPhe Glu Leu Asn Phe Glu Val Asn Ala Phe Val Tyr Asp Glu Asn 435 440 445Leu Ala Lys Asp Leu Arg Val Ala Tyr Glu His Asp Ile Thr Lys Ser 450 455460 Lys Gln Leu Thr Lys Glu Ser Tyr Ala Asn Arg Pro Leu Ser Val Lys 465470 475 480 Phe Lys Glu Ser Leu Ala Lys Leu Val Ser Pro Ile Leu 485 49019 420 DNA Staphylococcus aureus 19 atgaagattt tattcgtttg tacaggtaacacatgtcgta gcccattagc ggaaagtatt 60 gcaaaagagg ttatgccaaa tcatcaatttgaatcaagag gtatattcgc tgtgaacaat 120 caaggtgttt cgaattatgt tgaagacttagttgaagaac atcatttagc tgaaacgacc 180 ttatcgcaac aatttactga agcagatttgaaagcagata ttattttgac gatgtcgtat 240 tcgcacaaag aattaataga ggcacactttggtttgcaaa atcatgtttt cacattgcat 300 gaatatgtaa aagaagcagg agaagttatagatccatacg gtggaacaaa agaaatgtat 360 gtacatacct atgaagaact tgtaagtttaattttaaaat taaaagatat tatttgctag 420 20 139 PRT Staphylococcus aureus 20Met Lys Ile Leu Phe Val Cys Thr Gly Asn Thr Cys Arg Ser Pro Leu 1 5 1015 Ala Glu Ser Ile Ala Lys Glu Val Met Pro Asn His Gln Phe Glu Ser 20 2530 Arg Gly Ile Phe Ala Val Asn Asn Gln Gly Val Ser Asn Tyr Val Glu 35 4045 Asp Leu Val Glu Glu His His Leu Ala Glu Thr Thr Leu Ser Gln Gln 50 5560 Phe Thr Glu Ala Asp Leu Lys Ala Asp Ile Ile Leu Thr Met Ser Tyr 65 7075 80 Ser His Lys Glu Leu Ile Glu Ala His Phe Gly Leu Gln Asn His Val 8590 95 Phe Thr Leu His Glu Tyr Val Lys Glu Ala Gly Glu Val Ile Asp Pro100 105 110 Tyr Gly Gly Thr Lys Glu Met Tyr Val His Thr Tyr Glu Glu LeuVal 115 120 125 Ser Leu Ile Leu Lys Leu Lys Asp Ile Ile Cys 130 135 21837 DNA Staphylococcus aureus 21 atgacaaaac agattatagt aacagactcaacatccgatt tatctaaaga atacttagaa 60 gcaaacaaca ttcatgtaat tcctttaagtttaactattg aaggagcttc atacgttgac 120 caagtagata ttacatcaga agaatttattaatcatattg aaaatgatga agatgtaaag 180 acaagtcagc cagccatagg tgaatttatatctgcttatg aagaactagg aaaagatggc 240 tctgaaatca taagtattca tctttcttcaggattaagtg gtacatataa cactgcttac 300 caagcaagtc aaatggtaga tgctaatgtaactgttattg attcaaaatc tatttctttt 360 ggtttagggt atcaaataca acacctagtagagcttgtaa aagaaggtgt ctcaacttct 420 gaaatagtta aaaagttaaa tcatttaagagaaaacatta aattatttgt agttataggg 480 caattgaatc aattaattaa aggtggcagaattagtaaaa caaaaggttt gattggtaat 540 cttatgaaaa ttaaaccaat tggtacactagatgatggtc gcttagagct tgtgcacaat 600 gcgagaactc aaaattctag tatccaatacttgaaaaagg aaattgctga atttatagga 660 gatcatgaaa tcaaatccat tggtgtcgcacatgctaacg tcattgaata tgttgataaa 720 ttgaagaaag tttttaatga agcttttcatgtgaataatt acgatataaa tgtaactaca 780 ccagttattt ctgcacatac tggtcaaggtgcgattggcc tcgtagtcct taagaag 837 22 279 PRT Staphylococcus aureus 22Met Thr Lys Gln Ile Ile Val Thr Asp Ser Thr Ser Asp Leu Ser Lys 1 5 1015 Glu Tyr Leu Glu Ala Asn Asn Ile His Val Ile Pro Leu Ser Leu Thr 20 2530 Ile Glu Gly Ala Ser Tyr Val Asp Gln Val Asp Ile Thr Ser Glu Glu 35 4045 Phe Ile Asn His Ile Glu Asn Asp Glu Asp Val Lys Thr Ser Gln Pro 50 5560 Ala Ile Gly Glu Phe Ile Ser Ala Tyr Glu Glu Leu Gly Lys Asp Gly 65 7075 80 Ser Glu Ile Ile Ser Ile His Leu Ser Ser Gly Leu Ser Gly Thr Tyr 8590 95 Asn Thr Ala Tyr Gln Ala Ser Gln Met Val Asp Ala Asn Val Thr Val100 105 110 Ile Asp Ser Lys Ser Ile Ser Phe Gly Leu Gly Tyr Gln Ile GlnHis 115 120 125 Leu Val Glu Leu Val Lys Glu Gly Val Ser Thr Ser Glu IleVal Lys 130 135 140 Lys Leu Asn His Leu Arg Glu Asn Ile Lys Leu Phe ValVal Ile Gly 145 150 155 160 Gln Leu Asn Gln Leu Ile Lys Gly Gly Arg IleSer Lys Thr Lys Gly 165 170 175 Leu Ile Gly Asn Leu Met Lys Ile Lys ProIle Gly Thr Leu Asp Asp 180 185 190 Gly Arg Leu Glu Leu Val His Asn AlaArg Thr Gln Asn Ser Ser Ile 195 200 205 Gln Tyr Leu Lys Lys Glu Ile AlaGlu Phe Ile Gly Asp His Glu Ile 210 215 220 Lys Ser Ile Gly Val Ala HisAla Asn Val Ile Glu Tyr Val Asp Lys 225 230 235 240 Leu Lys Lys Val PheAsn Glu Ala Phe His Val Asn Asn Tyr Asp Ile 245 250 255 Asn Val Thr ThrPro Val Ile Ser Ala His Thr Gly Gln Gly Ala Ile 260 265 270 Gly Leu ValVal Leu Lys Lys 275 23 594 DNA Staphylococcus aureus 23 atgaatttattttacaatcc taaatatgta ggagatgtcg catttttaca aattgaacca 60 gttgaaggtgaattaaacta caataaaaaa ggtaatgttg ttgaaattac taatgaaggt 120 aatgttgtaggttataatat ttttgaaatt tcaaaagata taacaattga agaaaaaggt 180 catattaaattaactgatga acttgtaaat gtattccaaa agcgtatttc agaagctggt 240 tttgattataaattaaatgc tgatctatca ccgaaatttg tagttggcta cgttgaaact 300 aaagacaaacatcctgatgc agataaatta agtgtactaa atgtaaacgt tggaaatgac 360 acattacaaattgtatgtgg cgcgcctaac gttgaagctg gacagaaagt tgttgttgct 420 aaagtaggtgcagtgatgcc tagcggtatg gtaattaaag atgctgaatt acgtggtgtt 480 gcctcaagcggtatgatttg ttcaatgaaa gaattgaatt tacctaatgc acctgaagaa 540 aaaggtattatggtattaaa tgacagctat gaaattggac aagcattttt tgaa 594 24 198 PRTStaphylococcus aureus 24 Met Asn Leu Phe Tyr Asn Pro Lys Tyr Val Gly AspVal Ala Phe Leu 1 5 10 15 Gln Ile Glu Pro Val Glu Gly Glu Leu Asn TyrAsn Lys Lys Gly Asn 20 25 30 Val Val Glu Ile Thr Asn Glu Gly Asn Val ValGly Tyr Asn Ile Phe 35 40 45 Glu Ile Ser Lys Asp Ile Thr Ile Glu Glu LysGly His Ile Lys Leu 50 55 60 Thr Asp Glu Leu Val Asn Val Phe Gln Lys ArgIle Ser Glu Ala Gly 65 70 75 80 Phe Asp Tyr Lys Leu Asn Ala Asp Leu SerPro Lys Phe Val Val Gly 85 90 95 Tyr Val Glu Thr Lys Asp Lys His Pro AspAla Asp Lys Leu Ser Val 100 105 110 Leu Asn Val Asn Val Gly Asn Asp ThrLeu Gln Ile Val Cys Gly Ala 115 120 125 Pro Asn Val Glu Ala Gly Gln LysVal Val Val Ala Lys Val Gly Ala 130 135 140 Val Met Pro Ser Gly Met ValIle Lys Asp Ala Glu Leu Arg Gly Val 145 150 155 160 Ala Ser Ser Gly MetIle Cys Ser Met Lys Glu Leu Asn Leu Pro Asn 165 170 175 Ala Pro Glu GluLys Gly Ile Met Val Leu Asn Asp Ser Tyr Glu Ile 180 185 190 Gly Gln AlaPhe Phe Glu 195 25 717 DNA Staphylococcus aureus 25 atgactgtagaatggttagc agaacaatta aaagaacata atattcaatt aactgagact 60 caaaaacaacagtttcaaac atattatcgt ttacttgttg aatggaatga aaagatgaat 120 ttgacaagtattacagatga acacgatgta tatttgaaac atttttatga ttccattgca 180 cctagtttttattttgattt taatcagcct ataagtatat gtgatgtagg cgctggagct 240 ggttttccaagtattccgtt aaaaataatg tttccgcagt taaaagtgac gattgttgat 300 tcattaaataagcgtattca atttttaaac catttagcgt cagaattaca attacaggat 360 gtcagctttatacacgatag agcagaaaca tttggtaagg gtgtctacag ggagtcttat 420 gatgttgttactgcaagagc agtagctaga ttatccgtgt taagtgaatt gtgtttaccg 480 ctagttaaaaaaggtggaca gtttgttgca ttaaaatctt caaaaggtga agaagaatta 540 gaagaagcaaaatttgcaat tagtgtgtta ggtggtaatg ttacagaaac acataccttt 600 gaattgccagaagatgctgg agagcgccag atgttcatta ttgataaaaa aagacagacg 660 ccgaaaaagtatccaagaaa accagggacg cctaataaga ctcctttact tgaaaaa 717 26 239 PRTStaphylococcus aureus 26 Met Thr Val Glu Trp Leu Ala Glu Gln Leu Lys GluHis Asn Ile Gln 1 5 10 15 Leu Thr Glu Thr Gln Lys Gln Gln Phe Gln ThrTyr Tyr Arg Leu Leu 20 25 30 Val Glu Trp Asn Glu Lys Met Asn Leu Thr SerIle Thr Asp Glu His 35 40 45 Asp Val Tyr Leu Lys His Phe Tyr Asp Ser IleAla Pro Ser Phe Tyr 50 55 60 Phe Asp Phe Asn Gln Pro Ile Ser Ile Cys AspVal Gly Ala Gly Ala 65 70 75 80 Gly Phe Pro Ser Ile Pro Leu Lys Ile MetPhe Pro Gln Leu Lys Val 85 90 95 Thr Ile Val Asp Ser Leu Asn Lys Arg IleGln Phe Leu Asn His Leu 100 105 110 Ala Ser Glu Leu Gln Leu Gln Asp ValSer Phe Ile His Asp Arg Ala 115 120 125 Glu Thr Phe Gly Lys Gly Val TyrArg Glu Ser Tyr Asp Val Val Thr 130 135 140 Ala Arg Ala Val Ala Arg LeuSer Val Leu Ser Glu Leu Cys Leu Pro 145 150 155 160 Leu Val Lys Lys GlyGly Gln Phe Val Ala Leu Lys Ser Ser Lys Gly 165 170 175 Glu Glu Glu LeuGlu Glu Ala Lys Phe Ala Ile Ser Val Leu Gly Gly 180 185 190 Asn Val ThrGlu Thr His Thr Phe Glu Leu Pro Glu Asp Ala Gly Glu 195 200 205 Arg GlnMet Phe Ile Ile Asp Lys Lys Arg Gln Thr Pro Lys Lys Tyr 210 215 220 ProArg Lys Pro Gly Thr Pro Asn Lys Thr Pro Leu Leu Glu Lys 225 230 235 272358 DNA Staphylococcus aureus 27 atgataatat attggtgtat gacagttaatggagggaacg aaatgaaagc tttattactt 60 aaaacaagtg tatggctcgt tttgctttttagtgtaatgg gattatggca agtctcgaac 120 gcggctgagc agcatacacc aatgaaagcacatgcagtaa caacgataga caaagcaaca 180 acagataagc aacaagtacc gccaacaaaggaagcggctc atcattctgg caaagaagcg 240 gcaaccaacg tatcagcatc agcgcagggaacagctgatg atacaaacag caaagtaaca 300 tccaacgcac catctaacaa accatctacagtagtttcaa caaaagtaaa cgaaacacgc 360 gacgtagata cacaacaagc ctcaacacaaaaaccaactc acacagcaac gttcaaatta 420 tcaaatgcta aaacagcatc actttcaccacgaatgtttg ctgctaatgc accacaaaca 480 acaacacata aaatattaca tacaaatgatatccatggcc gactagccga agaaaaaggg 540 cgtgtcatcg gtatggctaa attaaaaacagtaaaagaac aagaaaagcc tgatttaatg 600 ttagacgcag gagacgcctt ccaaggtttaccactttcaa accagtctaa aggtgaagaa 660 atggctaaag caatgaatgc agtaggttatgatgctatgg cagtcggtaa ccatgaattt 720 gactttggat acgatcagtt gaaaaagttagagggtatgt tagacttccc gatgctaagt 780 actaacgttt ataaagatgg aaaacgcgcgtttaagcctt caacgattgt aacaaaaaat 840 ggtattcgtt atggaattat tggtgtaacgacaccagaaa caaagacgaa aacaagacct 900 gaaggcatta aaggcgttga atttagagatccattacaaa gtgtgacagc ggaaatgatg 960 cgtatttata aagacgtaga tacatttgttgttatatcac atttaggaat tgatccttca 1020 acacaagaaa catggcgtgg tgattacttagtgaaacaat taagtcaaaa tccacaattg 1080 aagaaacgta ttacagttat tgatggtcattcacatacag tacttcaaaa tggtcaaatt 1140 tataacaatg atgcattggc acaaacaggtacagcacttg cgaatatcgg taagattaca 1200 tttaattatc gcaatggaga ggtatcgaatattaaaccgt cattgattaa tgttaaagac 1260 gttgaaaatg taacaccgaa caaagcattagctgaacaaa ttaatcaagc tgatcaaaca 1320 tttagagcac aaactgcaga ggtaattattccaaacaata ccattgattt caaaggagaa 1380 agagatgacg ttagaacgcg tgaaacaaatttaggaaacg cgattgcaga tgctatggaa 1440 gcgtatggcg ttaagaattt ctctaaaaagactgactttg ccgtgacaaa tggtggaggt 1500 attcgtgcct ctatcgcaaa aggtaaggtgacacgctatg atttaatctc agtattacca 1560 tttggaaata cgattgcgca aattgatgtaaaaggttcag acgtctggac ggctttcgaa 1620 catagtttag gcgcaccaac aacacaaaaggacggtaaga cagtgttaac agcgaatggc 1680 ggtttactac atatctctga ttcaatccgtgtttactatg atataaataa accgtctggc 1740 aaacgaatta atgctattca aattttaaataaagagacag gtaagtttga aaatattgat 1800 ttaaaacgtg tatatcacgt aacgatgaatgacttcacag catcaggtgg cgacggatat 1860 agtatgttcg gtggtcctag agaagaaggtatttcattag atcaagtact agcaagttat 1920 ttaaaaacag ctaacttagc taagtatgatacgacagaac cacaacgtat gttattaggt 1980 aaaccagcag taagtgaaca accagctaaaggacaacaag gtagcaaagg tagtaagtct 2040 ggtaaagata cacaaccaat tggtgacgacaaagtgatgg atccagcgaa aaaaccagct 2100 ccaggtaaag ttgtattgtt gctagcgcatagaggaactg ttagtagcgg tacagaaggt 2160 tctggtcgca caatagaagg agctactgtatcaagcaaga gtgggaaaca attggctaga 2220 atgtcagtgc ctaaaggtag cgcgcatgagaaacagttac caaaaactgg aactaatcaa 2280 agttcaagcc cagaagcgat gtttgtattattagcaggta taggtttaat cgcgactgta 2340 cgacgtagaa aagctagc 2358 28 786PRT Staphylococcus aureus 28 Met Ile Ile Tyr Trp Cys Met Thr Val Asn GlyGly Asn Glu Met Lys 1 5 10 15 Ala Leu Leu Leu Lys Thr Ser Val Trp LeuVal Leu Leu Phe Ser Val 20 25 30 Met Gly Leu Trp Gln Val Ser Asn Ala AlaGlu Gln His Thr Pro Met 35 40 45 Lys Ala His Ala Val Thr Thr Ile Asp LysAla Thr Thr Asp Lys Gln 50 55 60 Gln Val Pro Pro Thr Lys Glu Ala Ala HisHis Ser Gly Lys Glu Ala 65 70 75 80 Ala Thr Asn Val Ser Ala Ser Ala GlnGly Thr Ala Asp Asp Thr Asn 85 90 95 Ser Lys Val Thr Ser Asn Ala Pro SerAsn Lys Pro Ser Thr Val Val 100 105 110 Ser Thr Lys Val Asn Glu Thr ArgAsp Val Asp Thr Gln Gln Ala Ser 115 120 125 Thr Gln Lys Pro Thr His ThrAla Thr Phe Lys Leu Ser Asn Ala Lys 130 135 140 Thr Ala Ser Leu Ser ProArg Met Phe Ala Ala Asn Ala Pro Gln Thr 145 150 155 160 Thr Thr His LysIle Leu His Thr Asn Asp Ile His Gly Arg Leu Ala 165 170 175 Glu Glu LysGly Arg Val Ile Gly Met Ala Lys Leu Lys Thr Val Lys 180 185 190 Glu GlnGlu Lys Pro Asp Leu Met Leu Asp Ala Gly Asp Ala Phe Gln 195 200 205 GlyLeu Pro Leu Ser Asn Gln Ser Lys Gly Glu Glu Met Ala Lys Ala 210 215 220Met Asn Ala Val Gly Tyr Asp Ala Met Ala Val Gly Asn His Glu Phe 225 230235 240 Asp Phe Gly Tyr Asp Gln Leu Lys Lys Leu Glu Gly Met Leu Asp Phe245 250 255 Pro Met Leu Ser Thr Asn Val Tyr Lys Asp Gly Lys Arg Ala PheLys 260 265 270 Pro Ser Thr Ile Val Thr Lys Asn Gly Ile Arg Tyr Gly IleIle Gly 275 280 285 Val Thr Thr Pro Glu Thr Lys Thr Lys Thr Arg Pro GluGly Ile Lys 290 295 300 Gly Val Glu Phe Arg Asp Pro Leu Gln Ser Val ThrAla Glu Met Met 305 310 315 320 Arg Ile Tyr Lys Asp Val Asp Thr Phe ValVal Ile Ser His Leu Gly 325 330 335 Ile Asp Pro Ser Thr Gln Glu Thr TrpArg Gly Asp Tyr Leu Val Lys 340 345 350 Gln Leu Ser Gln Asn Pro Gln LeuLys Lys Arg Ile Thr Val Ile Asp 355 360 365 Gly His Ser His Thr Val LeuGln Asn Gly Gln Ile Tyr Asn Asn Asp 370 375 380 Ala Leu Ala Gln Thr GlyThr Ala Leu Ala Asn Ile Gly Lys Ile Thr 385 390 395 400 Phe Asn Tyr ArgAsn Gly Glu Val Ser Asn Ile Lys Pro Ser Leu Ile 405 410 415 Asn Val LysAsp Val Glu Asn Val Thr Pro Asn Lys Ala Leu Ala Glu 420 425 430 Gln IleAsn Gln Ala Asp Gln Thr Phe Arg Ala Gln Thr Ala Glu Val 435 440 445 IleIle Pro Asn Asn Thr Ile Asp Phe Lys Gly Glu Arg Asp Asp Val 450 455 460Arg Thr Arg Glu Thr Asn Leu Gly Asn Ala Ile Ala Asp Ala Met Glu 465 470475 480 Ala Tyr Gly Val Lys Asn Phe Ser Lys Lys Thr Asp Phe Ala Val Thr485 490 495 Asn Gly Gly Gly Ile Arg Ala Ser Ile Ala Lys Gly Lys Val ThrArg 500 505 510 Tyr Asp Leu Ile Ser Val Leu Pro Phe Gly Asn Thr Ile AlaGln Ile 515 520 525 Asp Val Lys Gly Ser Asp Val Trp Thr Ala Phe Glu HisSer Leu Gly 530 535 540 Ala Pro Thr Thr Gln Lys Asp Gly Lys Thr Val LeuThr Ala Asn Gly 545 550 555 560 Gly Leu Leu His Ile Ser Asp Ser Ile ArgVal Tyr Tyr Asp Ile Asn 565 570 575 Lys Pro Ser Gly Lys Arg Ile Asn AlaIle Gln Ile Leu Asn Lys Glu 580 585 590 Thr Gly Lys Phe Glu Asn Ile AspLeu Lys Arg Val Tyr His Val Thr 595 600 605 Met Asn Asp Phe Thr Ala SerGly Gly Asp Gly Tyr Ser Met Phe Gly 610 615 620 Gly Pro Arg Glu Glu GlyIle Ser Leu Asp Gln Val Leu Ala Ser Tyr 625 630 635 640 Leu Lys Thr AlaAsn Leu Ala Lys Tyr Asp Thr Thr Glu Pro Gln Arg 645 650 655 Met Leu LeuGly Lys Pro Ala Val Ser Glu Gln Pro Ala Lys Gly Gln 660 665 670 Gln GlySer Lys Gly Ser Lys Ser Gly Lys Asp Thr Gln Pro Ile Gly 675 680 685 AspAsp Lys Val Met Asp Pro Ala Lys Lys Pro Ala Pro Gly Lys Val 690 695 700Val Leu Leu Leu Ala His Arg Gly Thr Val Ser Ser Gly Thr Glu Gly 705 710715 720 Ser Gly Arg Thr Ile Glu Gly Ala Thr Val Ser Ser Lys Ser Gly Lys725 730 735 Gln Leu Ala Arg Met Ser Val Pro Lys Gly Ser Ala His Glu LysGln 740 745 750 Leu Pro Lys Thr Gly Thr Asn Gln Ser Ser Ser Pro Glu AlaMet Phe 755 760 765 Val Leu Leu Ala Gly Ile Gly Leu Ile Ala Thr Val ArgArg Arg Lys 770 775 780 Ala Ser 785 29 609 DNA Staphylococcus aureus 29atgagtcatt attacgatga agatccaagt gtaattagca atgaacaacg tattcaatat 60caattaaacc atcataaaat tgatttaata actgataacg gagtgttttc gaaagataaa 120gtagattatg gttcagatgt tcttgttcaa acttttttaa aagcgcatcc acctggtcca 180agtaagcgaa ttgccgatgt tggttgtggt tacggaccaa ttggtttgat gattgctaaa 240gtatcaccac atcattcaat tacaatgcta gatgttaatc acagagcgct agccttagtt 300gaaaaaaaca aaaaattaaa tggtattgat aatgtgatcg taaaggaaag tgatgctttg 360tctgctgtgg aagacaaaag ttttgatttt attttaacca atccaccaat aagagcaggg 420aaagaaaccg tgcatcgtat attcgagcaa gcattacata gattagactc gaacggtgaa 480ctattcgttg taattcagaa gaagcaaggt atgccatctg caaagaaaag aatgaatgaa 540ctttttggaa atgtagaagt ggtaaataaa gataaaggat attacattct gagaagtata 600aaagcttga 609 30 202 PRT Staphylococcus aureus 30 Met Ser His Tyr TyrAsp Glu Asp Pro Ser Val Ile Ser Asn Glu Gln 1 5 10 15 Arg Ile Gln TyrGln Leu Asn His His Lys Ile Asp Leu Ile Thr Asp 20 25 30 Asn Gly Val PheSer Lys Asp Lys Val Asp Tyr Gly Ser Asp Val Leu 35 40 45 Val Gln Thr PheLeu Lys Ala His Pro Pro Gly Pro Ser Lys Arg Ile 50 55 60 Ala Asp Val GlyCys Gly Tyr Gly Pro Ile Gly Leu Met Ile Ala Lys 65 70 75 80 Val Ser ProHis His Ser Ile Thr Met Leu Asp Val Asn His Arg Ala 85 90 95 Leu Ala LeuVal Glu Lys Asn Lys Lys Leu Asn Gly Ile Asp Asn Val 100 105 110 Ile ValLys Glu Ser Asp Ala Leu Ser Ala Val Glu Asp Lys Ser Phe 115 120 125 AspPhe Ile Leu Thr Asn Pro Pro Ile Arg Ala Gly Lys Glu Thr Val 130 135 140His Arg Ile Phe Glu Gln Ala Leu His Arg Leu Asp Ser Asn Gly Glu 145 150155 160 Leu Phe Val Val Ile Arg Arg Lys Gln Gly Met Pro Ser Ala Lys Lys165 170 175 Arg Met Asn Glu Leu Phe Gly Asn Val Glu Val Val Asn Lys AspLys 180 185 190 Gly Tyr Tyr Ile Leu Arg Ser Ile Lys Ala 195 200 31 750DNA Staphylococcus aureus 31 atgcgagttg atttgaattg tgatttaggc gaagcatttggaaattattc ctttggtggt 60 gatcatcaaa ttattccgtt aattacaagt gcgaatgttgcttgtggttt tcacgctggt 120 gatgaaaatg taatgaatga aacggtaaaa cttgccaaagcacataatgt tgcagtaggt 180 gcacatcctg gtttacctga tttgaaaggc tttggcagacgaaatataga tatctctaac 240 gacgagattt ataatttgat gatttatcaa ttaggtgcattacaagggtt ttgtcgcatt 300 catcaagtta aaattaatca tgttaaaccg catggtgcattgtatcagat gggtgcaaaa 360 gacagagaaa tagcaaacgt tatagcacaa gctgtttatgactttgatcc atcactagtg 420 ttagtaggat tagcaaattc atatctaatt tcagaagcaaagaatgtcgg attaattaca 480 gcttctgaag tgtttgctga tagacgatac gaagatgatgggcagctcgt tagtagaaaa 540 gaaagtgatg ctgtgattac tgatactgac gaagcacttaagcaggtttt aaagatggtg 600 aaggaaaata aagttatttc aaaaaacaat aaggaagtaacgttacaagc agatacaatt 660 tgtgtgcatg gtgatggaga acatgcatta ttatttgtttcgaaaattag agaaatttta 720 atgaaagaag gcattgatat tcaatcctta 750 32 250PRT Staphylococcus aureus 32 Met Arg Val Asp Leu Asn Cys Asp Leu Gly GluAla Phe Gly Asn Tyr 1 5 10 15 Ser Phe Gly Gly Asp His Gln Ile Ile ProLeu Ile Thr Ser Ala Asn 20 25 30 Val Ala Cys Gly Phe His Ala Gly Asp GluAsn Val Met Asn Glu Thr 35 40 45 Val Lys Leu Ala Lys Ala His Asn Val AlaVal Gly Ala His Pro Gly 50 55 60 Leu Pro Asp Leu Lys Gly Phe Gly Arg ArgAsn Ile Asp Ile Ser Asn 65 70 75 80 Asp Glu Ile Tyr Asn Leu Met Ile TyrGln Leu Gly Ala Leu Gln Gly 85 90 95 Phe Cys Arg Ile His Gln Val Lys IleAsn His Val Lys Pro His Gly 100 105 110 Ala Leu Tyr Gln Met Gly Ala LysAsp Arg Glu Ile Ala Asn Val Ile 115 120 125 Ala Gln Ala Val Tyr Asp PheAsp Pro Ser Leu Val Leu Val Gly Leu 130 135 140 Ala Asn Ser Tyr Leu IleSer Glu Ala Lys Asn Val Gly Leu Ile Thr 145 150 155 160 Ala Ser Glu ValPhe Ala Asp Arg Arg Tyr Glu Asp Asp Gly Gln Leu 165 170 175 Val Ser ArgLys Glu Ser Asp Ala Val Ile Thr Asp Thr Asp Glu Ala 180 185 190 Leu LysGln Val Leu Lys Met Val Lys Glu Asn Lys Val Ile Ser Lys 195 200 205 AsnAsn Lys Glu Val Thr Leu Gln Ala Asp Thr Ile Cys Val His Gly 210 215 220Asp Gly Glu His Ala Leu Leu Phe Val Ser Lys Ile Arg Glu Ile Leu 225 230235 240 Met Lys Glu Gly Ile Asp Ile Gln Ser Leu 245 250 33 720 DNAStaphylococcus aureus 33 atggctcaaa tttctaaata taaacgtgta gttttgaaactaagtggtga agcgttagct 60 ggagaaaaag gatttggcat aaatccagta attattaaaagtgttgctga gcaagtggct 120 gaagttgcta aaatggactg tgaaatcgca gtaatcgttggtggcggaaa catttggaga 180 ggtaaaacag gtagtgactt aggtatggac cgtggaactgctgattacat gggtatgctt 240 gcaactgtaa tgaatgcctt agcattacaa gatagtttagaacaattgga ttgtgataca 300 cgagtattaa catctattga aatgaagcaa gtggctgaaccttatattcg tcgtcgtgca 360 attagacact tagaaaagaa acgcgtagtt atttttgctgcaggtattgg aaacccatac 420 ttctctacag atactacagc ggcattacgt gctgcagaagttgaagcaga tgttatttta 480 atgggcaaaa ataatgtaga tggtgtatat tctgcagatcctaaagtaaa caaagatgcg 540 gtaaaatatg aacatttaac gcatattcaa atgcttcaagaaggtttaca agtaatggat 600 tcaacagcat cctcattctg tatggataat aacattccgttaactgtttt ctctattatg 660 gaagaaggaa atattaaacg tgctgttatg ggtgaaaagataggtacgtt aattacaaaa 720 34 240 PRT Staphylococcus aureus 34 Met AlaGln Ile Ser Lys Tyr Lys Arg Val Val Leu Lys Leu Ser Gly 1 5 10 15 GluAla Leu Ala Gly Glu Lys Gly Phe Gly Ile Asn Pro Val Ile Ile 20 25 30 LysSer Val Ala Glu Gln Val Ala Glu Val Ala Lys Met Asp Cys Glu 35 40 45 IleAla Val Ile Val Gly Gly Gly Asn Ile Trp Arg Gly Lys Thr Gly 50 55 60 SerAsp Leu Gly Met Asp Arg Gly Thr Ala Asp Tyr Met Gly Met Leu 65 70 75 80Ala Thr Val Met Asn Ala Leu Ala Leu Gln Asp Ser Leu Glu Gln Leu 85 90 95Asp Cys Asp Thr Arg Val Leu Thr Ser Ile Glu Met Lys Gln Val Ala 100 105110 Glu Pro Tyr Ile Arg Arg Arg Ala Ile Arg His Leu Glu Lys Lys Arg 115120 125 Val Val Ile Phe Ala Ala Gly Ile Gly Asn Pro Tyr Phe Ser Thr Asp130 135 140 Thr Thr Ala Ala Leu Arg Ala Ala Glu Val Glu Ala Asp Val IleLeu 145 150 155 160 Met Gly Lys Asn Asn Val Asp Gly Val Tyr Ser Ala AspPro Lys Val 165 170 175 Asn Lys Asp Ala Val Lys Tyr Glu His Leu Thr HisIle Gln Met Leu 180 185 190 Gln Glu Gly Leu Gln Val Met Asp Ser Thr AlaSer Ser Phe Cys Met 195 200 205 Asp Asn Asn Ile Pro Leu Thr Val Phe SerIle Met Glu Glu Gly Asn 210 215 220 Ile Lys Arg Ala Val Met Gly Glu LysIle Gly Thr Leu Ile Thr Lys 225 230 235 240 35 33 DNA ArtificialSequence Oligonucleotide Primer 35 atatatctgc agtgataaat tgccaagcgt gac33 36 33 DNA Artificial Sequence Oligonucleotide Primer 36 atatatgagctctcttgtac agatttaggt ggc 33 37 33 DNA Artificial SequenceOligonucleotide Primer 37 atatatctgc agcaagtatt aggtgaagaa ggg 33 38 33DNA Artificial Sequence Oligonucleotide Primer 38 atatatgagc tcacggattgatcccaataa tgc 33 39 36 DNA Artificial Sequence Oligonucleotide Primer39 atatatctgc aggggattca aaaacaattt aacatc 36 40 36 DNA ArtificialSequence Oligonucleotide Primer 40 atatatgagc tcaaggctat taacttgcttatgatc 36 41 21 DNA Artificial Sequence Oligonucleotide Primer 41tggtgtgtac attgactgta c 21 42 21 DNA Artificial Sequence OligonucleotidePrimer 42 gctgttagtt cttgtgtttg g 21 43 33 DNA Artificial SequenceOligonucleotide Primer 43 atatatctgc agaggtattc accatgttac tgc 33 44 33DNA Artificial Sequence Oligonucleotide Primer 44 atatatgagc tcaattgatacacttggcca tcg 33 45 35 DNA Artificial Sequence Oligonucleotide Primer45 atatatctgc aggggacatt tttaatcatg catgc 35 46 36 DNA ArtificialSequence Oligonucleotide Primer 46 atatatgagc tcgcagtcat aataatagctaaagac 36 47 33 DNA Artificial Sequence Oligonucleotide Primer 47atatatctgc agtgttaatc gatacacatg tcc 33 48 33 DNA Artificial SequenceOligonucleotide Primer 48 atatatgagc tccttcaaac gcttagctaa agc 33 49 33DNA Artificial Sequence Oligonucleotide Primer 49 atatatctgc agacaagtgtatggctcgtt ttg 33 50 33 DNA Artificial Sequence Oligonucleotide Primer50 atatatgagc tcatttgaac gttgctgtgt gag 33 51 34 DNA Artificial SequenceOligonucleotide Primer 51 atatatctgc agagagtaca tactttcgct ttag 34 52 34DNA Artificial Sequence Oligonucleotide Primer 52 atatatgagc tccctaatcctagatattca tcac 34 53 33 DNA Artificial Sequence Oligonucleotide Primer53 atatatctgc agttgtacag gtaacacatg tcg 33 54 32 DNA Artificial SequenceOligonucleotide Primer 54 atatatgagc tcctgctttc aaatctgctc ag 32 55 21DNA Artificial Sequence Oligonucleotide Primer 55 ctattgaagg agcttcatacg 21 56 21 DNA Artificial Sequence Oligonucleotide Primer 56 tacaagctctactaggtgtt g 21 57 21 DNA Artificial Sequence Oligonucleotide Primer 57cctaaatatg taggagatgt c 21 58 21 DNA Artificial Sequence OligonucleotidePrimer 58 ttatctgcat caggatgttt g 21 59 21 DNA Artificial SequenceOligonucleotide Primer 59 ctgtagaatg gttagcagaa c 21 60 21 DNAArtificial Sequence Oligonucleotide Primer 60 caatcgtcac ttttaactgc g 2161 21 DNA Artificial Sequence Oligonucleotide Primer 61 acaagtgtatggctcgtttt g 21 62 21 DNA Artificial Sequence Oligonucleotide Primer 62atttgaacgt tgctgtgtga g 21 63 21 DNA Artificial Sequence OligonucleotidePrimer 63 attacgatga agatccaagt g 21 64 21 DNA Artificial SequenceOligonucleotide Primer 64 ttttttcaac taaggctagc g 21 65 21 DNAArtificial Sequence Oligonucleotide Primer 65 gaattgtgat ttaggcgaag c 2166 21 DNA Artificial Sequence Oligonucleotide Primer 66 atgaatgcgacaaaaccctt g 21 67 21 DNA Artificial Sequence Oligonucleotide Primer 67ttgaaactaa gtggtgaagc g 21 68 21 DNA Artificial Sequence OligonucleotidePrimer 68 gcagcaaaaa taactacgcg t 21 69 731 DNA Artificial SequenceNucleotide sequence of S. aureus coding region cloned for expression inE. col 69 atgggattaa agtatgaaca tattgctaag caacttaatg cgtttatacatcaatctaat 60 ttcaaacccg gtgataaatt gccaagcgtg acgcaattaa aagaacgttatcaagtaagt 120 aagagtacta tcattaaagc attaggctta ttggaacaag atggtttgatctatcaagca 180 caaggcagtg gtatttatgt gagaaatatt gctgatgcca atcgtatcaacgtctttaag 240 actaatggtt tctctaaaag tttaggtgaa caccgaatga caagtaaggtacttgttttt 300 aaggagattg caacgccacc taaatctgta caagatgagc tccaattaaatgcagatgat 360 accgtctact atttagagcg attaagattc gtggacgatg atgttttatgtatcgaatat 420 tcttattatc ataaagaaat cgtgaaatat ttaaatgatg atattgctaagggctctatc 480 ttcgactttt agaatcaaac atgaaacttc gtattggttt ttcagatattttctttaatg 540 tagatcaact cacttcaagt gaagcttcat tactacaatt gtctacaggtgaaccatgtt 600 tacgttacca ccagactttt tatacaatga ctggcaaacc ctttgattcatctgacatcg 660 tatttcatta tcgtcatgca cagttttata ttcctagtaa aaagagatctcatcaccatc 720 accatcacta a 731 70 243 PRT Artificial Sequence Aminoacid sequence encoded by S. aureus coding region cloned for expressionin E. col 70 Met Gly Leu Lys Tyr Glu His Ile Ala Lys Gln Leu Asn Ala PheIle 1 5 10 15 His Gln Ser Asn Phe Lys Pro Gly Asp Lys Leu Pro Ser ValThr Gln 20 25 30 Leu Lys Glu Arg Tyr Gln Val Ser Lys Ser Thr Ile Ile LysAla Leu 35 40 45 Gly Leu Leu Glu Gln Asp Gly Leu Ile Tyr Gln Ala Gln GlySer Gly 50 55 60 Ile Tyr Val Arg Asn Ile Ala Asp Ala Asn Arg Ile Asn ValPhe Lys 65 70 75 80 Thr Asn Gly Phe Ser Lys Ser Leu Gly Glu His Arg MetThr Ser Lys 85 90 95 Val Leu Val Phe Lys Glu Ile Ala Thr Pro Pro Lys SerVal Gln Asp 100 105 110 Glu Leu Gln Leu Asn Ala Asp Asp Thr Val Tyr TyrLeu Glu Arg Leu 115 120 125 Arg Phe Val Asp Asp Asp Val Leu Cys Ile GluTyr Ser Tyr Tyr His 130 135 140 Lys Glu Ile Val Lys Tyr Leu Asn Asp AspIle Ala Lys Gly Ser Ile 145 150 155 160 Phe Asp Tyr Leu Glu Ser Asn MetLys Leu Arg Ile Gly Phe Ser Asp 165 170 175 Ile Phe Phe Asn Val Asp GlnLeu Thr Ser Ser Glu Ala Ser Leu Leu 180 185 190 Gln Leu Ser Thr Gly GluPro Cys Leu Arg Tyr His Gln Thr Phe Tyr 195 200 205 Thr Met Thr Gly LysPro Phe Asp Ser Ser Asp Ile Val Phe His Tyr 210 215 220 Arg His Ala GlnPhe Tyr Ile Pro Ser Lys Lys Arg Ser His His His 225 230 235 240 His HisHis 71 855 DNA Artificial Sequence Nucleotide sequence of S. aureuscoding region cloned for expression in E. col 71 atgctggcac tttatggatttgcccaagga cttattcaag aagcaggaat tagaattaaa 60 caattgatgg agcaaaatttaacaattgaa acaaagtcaa atccgaatga ccttgttaca 120 aatgtagata aagcaacagaagatttcatt tttgatacaa ttttagaaac atatcccaat 180 catcaagtat taggtgaagaagggcatggt catgacatcg atacttccaa aggtacggta 240 tggattgttg acccaatagacggtacattg aattttgttc atcaacaaga aaatttcgca 300 atttcaattg gtatttatatcgatggtaaa ccttatgcag gttttgtata tgatgttatg 360 gctgatgtct tatatcatgctaaagtaggg gaaggtgcat atcgtggtag ccaacccttg 420 aaaccattga atgattctaatctaagacaa agcattattg ggatcaatcc gaactggtta 480 actaaaccaa ttttaggagaaatctttaaa gaaattgtta atgattctag aagtgcaagg 540 gcatatggta gtgcagcgcttgaaatcgtt tcagttgcta caggtaattt agaagcatat 600 atgacgccaa gacttcaaccatgggatttt gctggcggat tggttatttt atatgaagta 660 aatggacaag cttccaatttactaggagga ccattaacaa ttagtggtcc aaattcaatc 720 ttagttggaa atcgtggtctccatcaagaa attagcaatg attatttaga gccccaccat 780 gatgcgttaa tacaattacatgaacaacga tttaaaagaa aatcaaaaag atctcatcac 840 catcaccatc actaa 855 72284 PRT Artificial Sequence Amino acid sequence encoded by S. aureuscoding region cloned for expression in E. col 72 Met Leu Ala Leu Tyr GlyPhe Ala Gln Gly Leu Ile Gln Glu Ala Gly 1 5 10 15 Ile Arg Ile Lys GlnLeu Met Glu Gln Asn Leu Thr Ile Glu Thr Lys 20 25 30 Ser Asn Pro Asn AspLeu Val Thr Asn Val Asp Lys Ala Thr Glu Asp 35 40 45 Phe Ile Phe Asp ThrIle Leu Glu Thr Tyr Pro Asn His Gln Val Leu 50 55 60 Gly Glu Glu Gly HisGly His Asp Ile Asp Thr Ser Lys Gly Thr Val 65 70 75 80 Trp Ile Val AspPro Ile Asp Gly Thr Leu Asn Phe Val His Gln Gln 85 90 95 Glu Asn Phe AlaIle Ser Ile Gly Ile Tyr Ile Asp Gly Lys Pro Tyr 100 105 110 Ala Gly PheVal Tyr Asp Val Met Ala Asp Val Leu Tyr His Ala Lys 115 120 125 Val GlyGlu Gly Ala Tyr Arg Gly Ser Gln Pro Leu Lys Pro Leu Asn 130 135 140 AspSer Asn Leu Arg Gln Ser Ile Ile Gly Ile Asn Pro Asn Trp Leu 145 150 155160 Thr Lys Pro Ile Leu Gly Glu Ile Phe Lys Glu Ile Val Asn Asp Ser 165170 175 Arg Ser Ala Arg Ala Tyr Gly Ser Ala Ala Leu Glu Ile Val Ser Val180 185 190 Ala Thr Gly Asn Leu Glu Ala Tyr Met Thr Pro Arg Leu Gln ProTrp 195 200 205 Asp Phe Ala Gly Gly Leu Val Ile Leu Tyr Glu Val Asn GlyGln Ala 210 215 220 Ser Asn Leu Leu Gly Gly Pro Leu Thr Ile Ser Gly ProAsn Ser Ile 225 230 235 240 Leu Val Gly Asn Arg Gly Leu His Gln Glu IleSer Asn Asp Tyr Leu 245 250 255 Glu Pro His His Asp Ala Leu Ile Gln LeuHis Glu Gln Arg Phe Lys 260 265 270 Arg Lys Ser Lys Arg Ser His His HisHis His His 275 280 73 567 DNA Artificial Sequence Nucleotide sequenceof S. aureus coding region cloned for expression in E. col 73 atgggattcaaaaacaattt aacatcaaat ttaacaaata aaatcggtaa ttcagtcttt 60 aaaatagaaaatgttgacgg aaaaggtgca atgccaacga cgattcaaga attgagagaa 120 agacgacaacgtgctgaagc aattgtaaag agaaagtctt taatgtcatc aacaatgagc 180 gttgttccaattccgggttt agattttggt gttgatttaa aattaatgaa agatattatc 240 gaagatgttaataaaattta tggtttagat cataagcaag ttaatagcct tggggatgat 300 gtgaaagaaagaattatgtc tgcagcagca attcaaggta gtcaatttat tggtaaaaga 360 atttcaaatgcatttttaaa aattgtaatt agagatgtag ctaaacgtac tgctgcaaaa 420 caaacaaaatggtttcctgt tgtaggacaa gctgtgtctg catctattag ttactatttt 480 atgaataaaattggaaaaga tcacattcaa aaatgcgaaa atgttattaa aaatgtcatg 540 agatctcatcaccatcacca tcactaa 567 74 188 PRT Artificial Sequence Amino acidsequence encoded by S. aureus coding region cloned for expression in E.col 74 Met Gly Phe Lys Asn Asn Leu Thr Ser Asn Leu Thr Asn Lys Ile Gly 15 10 15 Asn Ser Val Phe Lys Ile Glu Asn Val Asp Gly Lys Gly Ala Met Pro20 25 30 Thr Thr Ile Gln Glu Leu Arg Glu Arg Arg Gln Arg Ala Glu Ala Ile35 40 45 Val Lys Arg Lys Ser Leu Met Ser Ser Thr Met Ser Val Val Pro Ile50 55 60 Pro Gly Leu Asp Phe Gly Val Asp Leu Lys Leu Met Lys Asp Ile Ile65 70 75 80 Glu Asp Val Asn Lys Ile Tyr Gly Leu Asp His Lys Gln Val AsnSer 85 90 95 Leu Gly Asp Asp Val Lys Glu Arg Ile Met Ser Ala Ala Ala IleGln 100 105 110 Gly Ser Gln Phe Ile Gly Lys Arg Ile Ser Asn Ala Phe LeuLys Ile 115 120 125 Val Ile Arg Asp Val Ala Lys Arg Thr Ala Ala Lys GlnThr Lys Trp 130 135 140 Phe Pro Val Val Gly Gln Ala Val Ser Ala Ser IleSer Tyr Tyr Phe 145 150 155 160 Met Asn Lys Ile Gly Lys Asp His Ile GlnLys Cys Glu Asn Val Ile 165 170 175 Lys Asn Val Met Arg Ser His His HisHis His His 180 185 75 996 DNA Artificial Sequence Nucleotide sequenceof S. aureus coding region cloned for expression in E. col 75 atgggaataaataatcatga attactaggt attcaccatg ttactgcaat gacagatgat 60 gcagaacgtaattataaatt ttttacagaa gtactaggca tgcgtttagt taaaaagaca 120 gtcaatcaagatgatattta tacgtatcat actttttttg cagatgatgt aggttcggca 180 ggtacagacatgacgttctt tgattttcca aatattacaa aagggcaggc aggaacaaat 240 tccattacaagaccgtcttt tagagtgcct aacgatgacg cattaacata ttatgaacag 300 cgctttgatgagtttggtgt taaacacgaa ggtattcaag aattatttgg taaaaaagtg 360 ttgccatttgaagaagtcga tggccaagtg tatcaattaa tttcagatga gttaaatgaa 420 ggggtagcacctggtgtacc ttggaagaat ggaccggttc cagtagataa agcgatttat 480 ggattaggccccattgaaat taaagtaagt tattttgacg actttaaaaa tattttagag 540 actgtttacggtatgacaac tattgcgcat gaagataatg tcgcattact tgaagttggc 600 gaaggaggcaatggtggcca ggtaatctta ataaaagatg ataaagggcc agcagcacgt 660 caaggttatggtgaggtaca tcatgtgtca tttcgtgtga aagatcatga tgcaatagaa 720 gcgtgggcaacgaaatataa agaggtaggt attaataact caggcatcgt taatcgtttc 780 tattttgaagcattatatgc acgtgtgggg catattttaa tagaaatttc aacagatgga 840 ccaggatttatggaagatga accttatgaa acattaggcg aagggttatc cttaccacca 900 tttttagaaaataaaagaga atatattgaa tcggaagtta gaccttttaa tacgaagcgt 960 caacatggtagatctcatca ccatcaccat cactaa 996 76 331 PRT Artificial Sequence Aminoacid sequence encoded by S. aureus coding region cloned for expressionin E. col 76 Met Gly Ile Asn Asn His Glu Leu Leu Gly Ile His His Val ThrAla 1 5 10 15 Met Thr Asp Asp Ala Glu Arg Asn Tyr Lys Phe Phe Thr GluVal Leu 20 25 30 Gly Met Arg Leu Val Lys Lys Thr Val Asn Gln Asp Asp IleTyr Thr 35 40 45 Tyr His Thr Phe Phe Ala Asp Asp Val Gly Ser Ala Gly ThrAsp Met 50 55 60 Thr Phe Phe Asp Phe Pro Asn Ile Thr Lys Gly Gln Ala GlyThr Asn 65 70 75 80 Ser Ile Thr Arg Pro Ser Phe Arg Val Pro Asn Asp AspAla Leu Thr 85 90 95 Tyr Tyr Glu Gln Arg Phe Asp Glu Phe Gly Val Lys HisGlu Gly Ile 100 105 110 Gln Glu Leu Phe Gly Lys Lys Val Leu Pro Phe GluGlu Val Asp Gly 115 120 125 Gln Val Tyr Gln Leu Ile Ser Asp Glu Leu AsnGlu Gly Val Ala Pro 130 135 140 Gly Val Pro Trp Lys Asn Gly Pro Val ProVal Asp Lys Ala Ile Tyr 145 150 155 160 Gly Leu Gly Pro Ile Glu Ile LysVal Ser Tyr Phe Asp Asp Phe Lys 165 170 175 Asn Ile Leu Glu Thr Val TyrGly Met Thr Thr Ile Ala His Glu Asp 180 185 190 Asn Val Ala Leu Leu GluVal Gly Glu Gly Gly Asn Gly Gly Gln Val 195 200 205 Ile Leu Ile Lys AspAsp Lys Gly Pro Ala Ala Arg Gln Gly Tyr Gly 210 215 220 Glu Val His HisVal Ser Phe Arg Val Lys Asp His Asp Ala Ile Glu 225 230 235 240 Ala TrpAla Thr Lys Tyr Lys Glu Val Gly Ile Asn Asn Ser Gly Ile 245 250 255 ValAsn Arg Phe Tyr Phe Glu Ala Leu Tyr Ala Arg Val Gly His Ile 260 265 270Leu Ile Glu Ile Ser Thr Asp Gly Pro Gly Phe Met Glu Asp Glu Pro 275 280285 Tyr Glu Thr Leu Gly Glu Gly Leu Ser Leu Pro Pro Phe Leu Glu Asn 290295 300 Lys Arg Glu Tyr Ile Glu Ser Glu Val Arg Pro Phe Asn Thr Lys Arg305 310 315 320 Gln His Gly Arg Ser His His His His His His 325 330 771113 DNA Artificial Sequence Nucleotide sequence of S. aureus codingregion cloned for expression in E. col 77 atgggacgta tcttaaaagagtccattatt gtggcatttg cctttgttgg tgttgtcgtt 60 ggtgccggct ttgctactggtcaagaaatt ttccagtttt tcacaagtca tggcgcatat 120 agcatttcag gcattattgtaacaggacta ttgattactt taggtggaat ggttgtcatg 180 catacaggtc atcatctaaagtccagaaat cattctgatt caattaacta tttcttatac 240 ccctctattg caagaggttttgatattatt ttaacaatgt ttatgttgtc tttagctatt 300 attatgactg caggtggtgcgtcaaccatt catcaaagtt tcaacttacc gtattggctg 360 agcgcactca tattagtcgcctttatttta gcaacactgt ttctaaaatt cgatcgttta 420 attgctgtgc ttggcggtgttaccccattt ttaattgcga ttgtcattat gattgcggtc 480 tactatttca caacaagtcatcttgatttt actgccgcta ataatgatgc tcagattcat 540 aagcagaaat cattatcacctggatggtgg tttgatgcga ttaactatgc aagcttgcaa 600 attgctgctg ccttcagcttcttatcagtg atgggtagta aagttaaata tcgtgactca 660 acgttatacg ggggcttgattggcggttta atcattacat ttttactcat gatgattaat 720 ctaggtttaa tttctcaattcgataaaatt aaacacgtag atctacctac attaaaatta 780 gcgacacaaa tgtctccgtcaattggtatt attatgtctg tcattatgat acttgtcatc 840 tacaatactg ttgttggattaatgtatgca tttgcgtcac gtttcagcgt tccattcagc 900 agacgttact tcatcattattattacaatg gctgtcatca cttatattag tacatttatc 960 ggtttcattt cattaattggaaaagtattc cctattatgg gattgttcgg tttcatctta 1020 ctcatacctg tactctataaaggtttaatt aagcgtatta ccggcaaatc tcatatcgat 1080 ggatccagat ctcatcaccatcaccatcac taa 1113 78 370 PRT Artificial Sequence Amino acid sequenceencoded by S. aureus coding region cloned for expression in E. col 78Met Gly Arg Ile Leu Lys Glu Ser Ile Ile Val Ala Phe Ala Phe Val 1 5 1015 Gly Val Val Val Gly Ala Gly Phe Ala Thr Gly Gln Glu Ile Phe Gln 20 2530 Phe Phe Thr Ser His Gly Ala Tyr Ser Ile Ser Gly Ile Ile Val Thr 35 4045 Gly Leu Leu Ile Thr Leu Gly Gly Met Val Val Met His Thr Gly His 50 5560 His Leu Lys Ser Arg Asn His Ser Asp Ser Ile Asn Tyr Phe Leu Tyr 65 7075 80 Pro Ser Ile Ala Arg Gly Phe Asp Ile Ile Leu Thr Met Phe Met Leu 8590 95 Ser Leu Ala Ile Ile Met Thr Ala Gly Gly Ala Ser Thr Ile His Gln100 105 110 Ser Phe Asn Leu Pro Tyr Trp Leu Ser Ala Leu Ile Leu Val AlaPhe 115 120 125 Ile Leu Ala Thr Leu Phe Leu Lys Phe Asp Arg Leu Ile AlaVal Leu 130 135 140 Gly Gly Val Thr Pro Phe Leu Ile Ala Ile Val Ile MetIle Ala Val 145 150 155 160 Tyr Tyr Phe Thr Thr Ser His Leu Asp Phe ThrAla Ala Asn Asn Asp 165 170 175 Ala Gln Ile His Lys Gln Lys Ser Leu SerPro Gly Trp Trp Phe Asp 180 185 190 Ala Ile Asn Tyr Ala Ser Leu Gln IleAla Ala Ala Phe Ser Phe Leu 195 200 205 Ser Val Met Gly Ser Lys Val LysTyr Arg Asp Ser Thr Leu Tyr Gly 210 215 220 Gly Leu Ile Gly Gly Leu IleIle Thr Phe Leu Leu Met Met Ile Asn 225 230 235 240 Leu Gly Leu Ile SerGln Phe Asp Lys Ile Lys His Val Asp Leu Pro 245 250 255 Thr Leu Lys LeuAla Thr Gln Met Ser Pro Ser Ile Gly Ile Ile Met 260 265 270 Ser Val IleMet Ile Leu Val Ile Tyr Asn Thr Val Val Gly Leu Met 275 280 285 Tyr AlaPhe Ala Ser Arg Phe Ser Val Pro Phe Ser Arg Arg Tyr Phe 290 295 300 IleIle Ile Ile Thr Met Ala Val Ile Thr Tyr Ile Ser Thr Phe Ile 305 310 315320 Gly Phe Ile Ser Leu Ile Gly Lys Val Phe Pro Ile Met Gly Leu Phe 325330 335 Gly Phe Ile Leu Leu Ile Pro Val Leu Tyr Lys Gly Leu Ile Lys Arg340 345 350 Ile Thr Gly Lys Ser His Ile Asp Gly Phe Arg Ser His His HisHis 355 360 365 His His 370 79 801 DNA Artificial Sequence Nucleotidesequence of S. aureus coding region cloned for expression in E. col 79atgggattaa tcgatacaca tgtccattta aatgatgagc aatacgatga tgatttgagt 60gaagtgatta cacgtgctag agaagcaggt gttgatcgta tgtttgtagt tggttttaac 120aaatcgacaa ttgaacgcgc gatgaaatta atcgatgagt atgatttttt atatggcatt 180atcggttggc atccagttga cgcaattgat tttacagaag aacacttgga atggattgaa 240tctttagctc agcatccaaa agtgattggt attggtgaaa tgggattaga ttatcactgg 300gataaatctc ctgcagatgt tcaaaaggaa gtttttagaa agcaaattgc tttagctaag 360cgtttgaagt taccaattat cattcataac cgtgaagcaa ctcaagactg tatcgatatc 420ttattggagg agcatgctga agaggtaggc gggattatgc atagctttag tggttctcca 480gaaattgcag atattgtaac taataagctg aatttttata tttcattagg tggacctgtg 540acatttaaaa atgctaaaca gcctaaagaa gttgctaagc atgtgtcaat ggagcgtttg 600ctagttgaaa ccgatgcacc gtatctttcg ccacatccgt atagagggaa gcgaaatgaa 660ccggcgagag taactttagt agctgaacaa attgctgaat taaaaggctt atcttatgaa 720gaagtgtgcg aacaaacaac taaaaatgca gagaaattgt ttaatttaaa ttcaagatct 780catcaccatc accatcacta a 801 80 266 PRT Artificial Sequence Amino acidsequence encoded by S. aureus coding region cloned for expression in E.col 80 Met Gly Leu Ile Asp Thr His Val His Leu Asn Asp Glu Gln Tyr Asp 15 10 15 Asp Asp Leu Ser Glu Val Ile Thr Arg Ala Arg Glu Ala Gly Val Asp20 25 30 Arg Met Phe Val Val Gly Phe Asn Lys Ser Thr Ile Glu Arg Ala Met35 40 45 Lys Leu Ile Asp Glu Tyr Asp Phe Leu Tyr Gly Ile Ile Gly Trp His50 55 60 Pro Val Asp Ala Ile Asp Phe Thr Glu Glu His Leu Glu Trp Ile Glu65 70 75 80 Ser Leu Ala Gln His Pro Lys Val Ile Gly Ile Gly Glu Met GlyLeu 85 90 95 Asp Tyr His Trp Asp Lys Ser Pro Ala Asp Val Gln Lys Glu ValPhe 100 105 110 Arg Lys Gln Ile Ala Leu Ala Lys Arg Leu Lys Leu Pro IleIle Ile 115 120 125 His Asn Arg Glu Ala Thr Gln Asp Cys Ile Asp Ile LeuLeu Glu Glu 130 135 140 His Ala Glu Glu Val Gly Gly Ile Met His Ser PheSer Gly Ser Pro 145 150 155 160 Glu Ile Ala Asp Ile Val Thr Asn Lys LeuAsn Phe Tyr Ile Ser Leu 165 170 175 Gly Gly Pro Val Thr Phe Lys Asn AlaLys Gln Pro Lys Glu Val Ala 180 185 190 Lys His Val Ser Met Glu Arg LeuLeu Val Glu Thr Asp Ala Pro Tyr 195 200 205 Leu Ser Pro His Pro Tyr ArgGly Lys Arg Asn Glu Pro Ala Arg Val 210 215 220 Thr Leu Val Ala Glu GlnIle Ala Glu Leu Lys Gly Leu Ser Tyr Glu 225 230 235 240 Glu Val Cys GluGln Thr Thr Lys Asn Ala Glu Lys Leu Phe Asn Leu 245 250 255 Asn Ser ArgSer His His His His His His 260 265 81 2150 DNA Artificial SequenceNucleotide sequence of S. aureus coding region cloned for expression inE. col 81 atgggaataa tatattggtg tatgacagtt aatggaggga acgaaatgaaagctttatta 60 cttaaaacaa gtgtatggct cgttttgctt tttagtgtaa tgggattatggcaagtctcg 120 aacgcggctg agcagcatac accaatgaaa gcacatgcag taacaacgatagacaaagca 180 acaacagata agcaacaagt accgccaaca aaggaagcgg ctcatcattctggcaaagaa 240 gcggcaacca acgtatcagc atcagcgcag ggaacagctg atgatacaaacagcaaagta 300 acatccaacg caccatctaa caaaccatct acagtagttt caacaaaagtaaacgaaaca 360 cgcgacgtag atacacaaca agcctcaaca caaaaaccaa ctcacacagcaacgttcaaa 420 ttatcaaatg ctaaaacagc atcactttca ccacgaatgt ttgctgctaatgcaccacaa 480 acaacaacac ataaaatatt acatacaaat gatatccatg gccgactagccgaagaaaaa 540 gggcgtgtca tcggtatggc taaattaaaa acagtaaaag aacaagaaaagcctgattta 600 atgttagacg caggagacgc cttccaaggt ttaccacttt caaaccagtctaaaggtgaa 660 gaaatggcta aagcaatgaa tgcagtaggt tatgatgcta tggcagtcggtaaccatgaa 720 tttgactttg gatacgatca gttgaaaaag ttagagggta tgttagacttcccgatgcta 780 agtactaacg tttataaaga tggaaaacgc gcgtttaagc cttcaacgattgtaacaaaa 840 aatggtattc gttatggaat tattggtgta acgacaccag aaacaaagacgaaaacaaga 900 cctgaaggca ttaaaggcgt tgaatttaga gatccattac aaagtgtgacagcggaaatg 960 atgcgtattt ataaagacgt agatacattt gttgttatat cacatttaggaattgatcct 1020 tcaacacaag aaacatggcg tggtgattac ttagtgaaac aattaagtcaaaatccacaa 1080 ttgaagaaac gtattacagt tattgatggt cattcacata cagtacttcaaaatggtcaa 1140 atttataaca atgatgcatt ggcacaaaca ggtacagcac ttgcgaatatcggtaagatt 1200 acatttaatt atcgcaatgg agaggtatcg aatattaaac cgtcattgattaatgttaaa 1260 gacgttgaaa atgtaacacc gaacaaagca ttagctgaac aaattaatcaagctgatcaa 1320 acatttagag cacaaactgc agaggtaatt attccaaaca ataccattgatttcaaagga 1380 gaaagagatg acgttagaac gcgtgaaaca aatttaggaa acgcgattgcagatgctatg 1440 gaagcgtatg gcgttaagaa tttctctaaa aagactgact ttgccgtgacaaatggtgga 1500 ggtattcgtg cctctatcgc aaaaggtaag gtgacacgct atgatttaatctcagtatta 1560 ccatttggaa atacgattgc gcaaattgat gtaaaaggtt cagacgtctggacggctttc 1620 gaacatagtt taggcgcacc aacaacacaa aaggacggta agacagtgttaacagcgaat 1680 ggcggtttac tacatatctc tgattcaatc cgtgtttact atgatataaataaaccgtct 1740 ggcaaacgaa ttaatgctat tcaaatttta aataaagaga caggtaagtttgaaaatatt 1800 gatttaaaac gtgtatatca cgtaacgatg aatgacttca cagcatcaggtgggacggat 1860 atagtatgtt cggtggtcct agagaagaag gtatttcatt agatcaagtactagcaagtt 1920 atttaaaaac agctaactta gctaagtatg atacgacaga accacaacgtatgttattag 1980 gtaaaccagc agtaagtgaa caaccagcta aaggacaaca aggtagcaaaggtagtaagt 2040 ctggtaaaga tacacaacca attggtgacg acaaagtgat ggatccagcgaaaaaaccag 2100 ctccaggtaa agttgtattg ttgagatctc atcaccatca ccatcactaa2150 82 716 PRT Artificial Sequence Amino acid sequence encoded by S.aureus coding region cloned for expression in E. col 82 Met Gly Ile IleTyr Trp Cys Met Thr Val Asn Gly Gly Asn Glu Met 1 5 10 15 Lys Ala LeuLeu Leu Lys Thr Ser Val Trp Leu Val Leu Leu Phe Ser 20 25 30 Val Met GlyLeu Trp Gln Val Ser Asn Ala Ala Glu Gln His Thr Pro 35 40 45 Met Lys AlaHis Ala Val Thr Thr Ile Asp Lys Ala Thr Thr Asp Lys 50 55 60 Gln Gln ValPro Pro Thr Lys Glu Ala Ala His His Ser Gly Lys Glu 65 70 75 80 Ala AlaThr Asn Val Ser Ala Ser Ala Gln Gly Thr Ala Asp Asp Thr 85 90 95 Asn SerLys Val Thr Ser Asn Ala Pro Ser Asn Lys Pro Ser Thr Val 100 105 110 ValSer Thr Lys Val Asn Glu Thr Arg Asp Val Asp Thr Gln Gln Ala 115 120 125Ser Thr Gln Lys Pro Thr His Thr Ala Thr Phe Lys Leu Ser Asn Ala 130 135140 Lys Thr Ala Ser Leu Ser Pro Arg Met Phe Ala Ala Asn Ala Pro Gln 145150 155 160 Thr Thr Thr His Lys Ile Leu His Thr Asn Asp Ile His Gly ArgLeu 165 170 175 Ala Glu Glu Lys Gly Arg Val Ile Gly Met Ala Lys Leu LysThr Val 180 185 190 Lys Glu Gln Glu Lys Pro Asp Leu Met Leu Asp Ala GlyAsp Ala Phe 195 200 205 Gln Gly Leu Pro Leu Ser Asn Gln Ser Lys Gly GluGlu Met Ala Lys 210 215 220 Ala Met Asn Ala Val Gly Tyr Asp Ala Met AlaVal Gly Asn His Glu 225 230 235 240 Phe Asp Phe Gly Tyr Asp Gln Leu LysLys Leu Glu Gly Met Leu Asp 245 250 255 Phe Pro Met Leu Ser Thr Asn ValTyr Lys Asp Gly Lys Arg Ala Phe 260 265 270 Lys Pro Ser Thr Ile Val ThrLys Asn Gly Ile Arg Tyr Gly Ile Ile 275 280 285 Gly Val Thr Thr Pro GluThr Lys Thr Lys Thr Arg Pro Glu Gly Ile 290 295 300 Lys Gly Val Glu PheArg Asp Pro Leu Gln Ser Val Thr Ala Glu Met 305 310 315 320 Met Arg IleTyr Lys Asp Val Asp Thr Phe Val Val Ile Ser His Leu 325 330 335 Gly IleAsp Pro Ser Thr Gln Glu Thr Trp Arg Gly Asp Tyr Leu Val 340 345 350 LysGln Leu Ser Gln Asn Pro Gln Leu Lys Lys Arg Ile Thr Val Ile 355 360 365Asp Gly His Ser His Thr Val Leu Gln Asn Gly Gln Ile Tyr Asn Asn 370 375380 Asp Ala Leu Ala Gln Thr Gly Thr Ala Leu Ala Asn Ile Gly Lys Ile 385390 395 400 Thr Phe Asn Tyr Arg Asn Gly Glu Val Ser Asn Ile Lys Pro SerLeu 405 410 415 Ile Asn Val Lys Asp Val Glu Asn Val Thr Pro Asn Lys AlaLeu Ala 420 425 430 Glu Gln Ile Asn Gln Ala Asp Gln Thr Phe Arg Ala GlnThr Ala Glu 435 440 445 Val Ile Ile Pro Asn Asn Thr Ile Asp Phe Lys GlyGlu Arg Asp Asp 450 455 460 Val Arg Thr Arg Glu Thr Asn Leu Gly Asn AlaIle Ala Asp Ala Met 465 470 475 480 Glu Ala Tyr Gly Val Lys Asn Phe SerLys Lys Thr Asp Phe Ala Val 485 490 495 Thr Asn Gly Gly Gly Ile Arg AlaSer Ile Ala Lys Gly Lys Val Thr 500 505 510 Arg Tyr Asp Leu Ile Ser ValLeu Pro Phe Gly Asn Thr Ile Ala Gln 515 520 525 Ile Asp Val Lys Gly SerAsp Val Trp Thr Ala Phe Glu His Ser Leu 530 535 540 Gly Ala Pro Thr ThrGln Lys Asp Gly Lys Thr Val Leu Thr Ala Asn 545 550 555 560 Gly Gly LeuLeu His Ile Ser Asp Ser Ile Arg Val Tyr Tyr Asp Ile 565 570 575 Asn LysPro Ser Gly Lys Arg Ile Asn Ala Ile Gln Ile Leu Asn Lys 580 585 590 GluThr Gly Lys Phe Glu Asn Ile Asp Leu Lys Arg Val Tyr His Val 595 600 605Thr Met Asn Asp Phe Thr Ala Ser Gly Gly Asp Gly Tyr Ser Met Phe 610 615620 Gly Gly Pro Arg Glu Glu Gly Ile Ser Leu Asp Gln Val Leu Ala Ser 625630 635 640 Tyr Leu Lys Thr Ala Asn Leu Ala Lys Tyr Asp Thr Thr Glu ProGln 645 650 655 Arg Met Leu Leu Gly Lys Pro Ala Val Ser Glu Gln Pro AlaLys Gly 660 665 670 Gln Gln Gly Ser Lys Gly Ser Lys Ser Gly Lys Asp ThrGln Pro Ile 675 680 685 Gly Asp Asp Lys Val Met Asp Pro Ala Lys Lys ProAla Pro Gly Lys 690 695 700 Val Val Leu Leu Arg Ser His His His His HisHis 705 710 715 83 1509 DNA Artificial Sequence Nucleotide sequence ofS. aureus coding region cloned for expression in E. col 83 atgggacgatttacattttc aaacgattta ggaacgttat ttactattat tttagccatt 60 ggattcatcattaatttagt attggctttt attattatct ttttagaaag aaataggcgt 120 acagcgagttcaacttgggc atggctattt gtactttttg tcttaccatt gattggtttt 180 attctttacttgttttttgg tagaaccgtt tcggcacgca aattgaataa aaacaatggt 240 aacgtgttaacggatttcga tggactttta aaacaacaaa tagaaagctt tgataaaggt 300 aattatggtactgataacaa acaagttcaa aaacatcatg atttagtacg tatgcttttg 360 atggatcaagatggtttttt aactgaaaat aataaagttg atcatttcat tgatggaaat 420 gatttatatgatcaagtttt aaaagatatt aaaaatgcaa aagaatatat ccatttagag 480 tactatactttcgctttaga tggtttaggt aaaagaattt tacatgcttt agaagaaaaa 540 ttgaaacaaggtctagaagt aaaaatatta tatgatgatg ttggatctaa aaatgttaag 600 atggcaaattttgatcattt taaatcgtta ggtggagaag ttgaagcatt ttttgcttca 660 aaattaccgttattgaattt cagaatgaat aatagaaatc atagaaaaat catcgtaatc 720 gatggtcaactaggttatgt cggaggattt aacattggtg atgaatatct aggattagga 780 aaattaggatattggagaga tacgcattta cgtatacaag gggatgcggt tgatgcactg 840 cagttgcgatttattttaga ctggaattcg caagcgcacc gtccacaatt tgaatatgat 900 gttaagtatttccctaaaaa gaacggacca ttgggcaatt caccaattca aatagctgca 960 agtggcccggctagtgactg gcatcaaatt gaatacggtt atacaaaaat gattatgagt 1020 gcaaagaaatctgtatattt acaatcacca tatttcattc cggataattc atatataaat 1080 gccattaaaattgctgctaa atcaggtgta gatgtacatt taatgattcc atgtaagcca 1140 gatcatccattagtatattg ggcgacattt tcaaatgcct ctgacttatt atcaagtggt 1200 gttaaaatttatacgtatga aaatggattt atacattcta aaatgtgctt aattgatgat 1260 gaaatcgtatcagtgggcac agcaaatatg gactttagaa gttttgaatt aaattttgaa 1320 gtaaatgcctttgtatatga tgaaaatctt gctaaagatt taagggtggc ttatgaacat 1380 gatattacaaaatcaaaaca actaaccaaa gaatcatatg ccaatagacc gctgtctgtt 1440 aaattcaaagaatcgttagc aaaattagtt tcgccaattt taagatctca tcaccatcac 1500 catcactaa1509 84 502 PRT Artificial Sequence Amino acid sequence encoded by S.aureus coding region cloned for expression in E. col 84 Met Gly Arg PheThr Phe Ser Asn Asp Leu Gly Thr Leu Phe Thr Ile 1 5 10 15 Ile Leu AlaIle Gly Phe Ile Ile Asn Leu Val Leu Ala Phe Ile Ile 20 25 30 Ile Phe LeuGlu Arg Asn Arg Arg Thr Ala Ser Ser Thr Trp Ala Trp 35 40 45 Leu Phe ValLeu Phe Val Leu Pro Leu Ile Gly Phe Ile Leu Tyr Leu 50 55 60 Phe Phe GlyArg Thr Val Ser Ala Arg Lys Leu Asn Lys Asn Asn Gly 65 70 75 80 Asn ValLeu Thr Asp Phe Asp Gly Leu Leu Lys Gln Gln Ile Glu Ser 85 90 95 Phe AspLys Gly Asn Tyr Gly Thr Asp Asn Lys Gln Val Gln Lys His 100 105 110 HisAsp Leu Val Arg Met Leu Leu Met Asp Gln Asp Gly Phe Leu Thr 115 120 125Glu Asn Asn Lys Val Asp His Phe Ile Asp Gly Asn Asp Leu Tyr Asp 130 135140 Gln Val Leu Lys Asp Ile Lys Asn Ala Lys Glu Tyr Ile His Leu Glu 145150 155 160 Tyr Tyr Thr Phe Ala Leu Asp Gly Leu Gly Lys Arg Ile Leu HisAla 165 170 175 Leu Glu Glu Lys Leu Lys Gln Gly Leu Glu Val Lys Ile LeuTyr Asp 180 185 190 Asp Val Gly Ser Lys Asn Val Lys Met Ala Asn Phe AspHis Phe Lys 195 200 205 Ser Leu Gly Gly Glu Val Glu Ala Phe Phe Ala SerLys Leu Pro Leu 210 215 220 Leu Asn Phe Arg Met Asn Asn Arg Asn His ArgLys Ile Ile Val Ile 225 230 235 240 Asp Gly Gln Leu Gly Tyr Val Gly GlyPhe Asn Ile Gly Asp Glu Tyr 245 250 255 Leu Gly Leu Gly Lys Leu Gly TyrTrp Arg Asp Thr His Leu Arg Ile 260 265 270 Gln Gly Asp Ala Val Asp AlaLeu Gln Leu Arg Phe Ile Leu Asp Trp 275 280 285 Asn Ser Gln Ala His ArgPro Gln Phe Glu Tyr Asp Val Lys Tyr Phe 290 295 300 Pro Lys Lys Asn GlyPro Leu Gly Asn Ser Pro Ile Gln Ile Ala Ala 305 310 315 320 Ser Gly ProAla Ser Asp Trp His Gln Ile Glu Tyr Gly Tyr Thr Lys 325 330 335 Met IleMet Ser Ala Lys Lys Ser Val Tyr Leu Gln Ser Pro Tyr Phe 340 345 350 IlePro Asp Asn Ser Tyr Ile Asn Ala Ile Lys Ile Ala Ala Lys Ser 355 360 365Gly Val Asp Val His Leu Met Ile Pro Cys Lys Pro Asp His Pro Leu 370 375380 Val Tyr Trp Ala Thr Phe Ser Asn Ala Ser Asp Leu Leu Ser Ser Gly 385390 395 400 Val Lys Ile Tyr Thr Tyr Glu Asn Gly Phe Ile His Ser Lys MetCys 405 410 415 Leu Ile Asp Asp Glu Ile Val Ser Val Gly Thr Ala Asn MetAsp Phe 420 425 430 Arg Ser Phe Glu Leu Asn Phe Glu Val Asn Ala Phe ValTyr Asp Glu 435 440 445 Asn Leu Ala Lys Asp Leu Arg Val Ala Tyr Glu HisAsp Ile Thr Lys 450 455 460 Ser Lys Gln Leu Thr Lys Glu Ser Tyr Ala AsnArg Pro Leu Ser Val 465 470 475 480 Lys Phe Lys Glu Ser Leu Ala Lys LeuVal Ser Pro Ile Leu Arg Ser 485 490 495 His His His His His His 500 85447 DNA Artificial Sequence Nucleotide sequence of S. aureus codingregion cloned for expression in E. col 85 atgggaaaga ttttattcgtttgtacaggt aacacatgtc gtagcccatt agcggaaagt 60 attgcaaaag aggttatgccaaatcatcaa tttgaatcaa gaggtatatt cgctgtgaac 120 aatcaaggtg tttcgaattatgttgaagac ttagttgaag aacatcattt agctgaaacg 180 accttatcgc aacaatttactgaagcagat ttgaaagcag atattatttt gacgatgtcg 240 tattcgcaca aagaattaatagaggcacac tttggtttgc aaaatcatgt tttcacattg 300 catgaatatg taaaagaagcaggagaagtt atagatccat acggtggaac aaaagaaatg 360 tatgtacata cctatgaagaacttgtaagt ttaattttaa aattaaaaga tattatttgc 420 agatctcatc accatcaccatcactaa 447 86 148 PRT Artificial Sequence Amino acid sequence encodedby S. aureus coding region cloned for expression in E. col 86 Met GlyLys Ile Leu Phe Val Cys Thr Gly Asn Thr Cys Arg Ser Pro 1 5 10 15 LeuAla Glu Ser Ile Ala Lys Glu Val Met Pro Asn His Gln Phe Glu 20 25 30 SerArg Gly Ile Phe Ala Val Asn Asn Gln Gly Val Ser Asn Tyr Val 35 40 45 GluAsp Leu Val Glu Glu His His Leu Ala Glu Thr Thr Leu Ser Gln 50 55 60 GlnPhe Thr Glu Ala Asp Leu Lys Ala Asp Ile Ile Leu Thr Met Ser 65 70 75 80Tyr Ser His Lys Glu Leu Ile Glu Ala His Phe Gly Leu Gln Asn His 85 90 95Val Phe Thr Leu His Glu Tyr Val Lys Glu Ala Gly Glu Val Ile Asp 100 105110 Pro Tyr Gly Gly Thr Lys Glu Met Tyr Val His Thr Tyr Glu Glu Leu 115120 125 Val Ser Leu Ile Leu Lys Leu Lys Asp Ile Ile Cys Arg Ser His His130 135 140 His His His His 145 87 744 DNA Staphylococcus aureus 87atggctcaaa tttctaaata taaacgtgta gttttgaaac taagtggtga agcgttagct 60ggagaaaaag gatttggcat aaatccagta attattaaaa gtgttgctga gcaagtggct 120gaagttgcta aaatggactg tgaaatcgca gtaatcgttg gtggcggaaa catttggaga 180ggtaaaacag gtagtgactt aggtatggac cgtggaactg ctgattacat gggtatgctt 240gcaactgtaa tgaatgcctt agcattacaa gatagtttag aacaattgga ttgtgataca 300cgagtattaa catctattga aatgaagcaa gtggctgaac cttatattcg tcgtcgtgca 360attagacact tagaaaagaa acgcgtagtt atttttgctg caggtattgg aaacccatac 420ttctctacag atactacagc ggcattacgt gctgcagaag ttgaagcaga tgttatttta 480atgggcaaaa ataatgtaga tggtgtatat tctgcagatc ctaaagtaaa caaagatgcg 540gtaaaatatg aacatttaac gcatattcaa atgcttcaag aaggtttaca agtaatggat 600tcaacagcat cctcattctg tatggataat aacattccgt taactgtttt ctctattatg 660gaagaaggaa atattaaacg tgctgttatg ggtgaaaaga taggtacgtt aattacaaaa 720agatctcatc accatcacca tcac 744 88 248 PRT Staphylococcus aureus 88 MetAla Gln Ile Ser Lys Tyr Lys Arg Val Val Leu Lys Leu Ser Gly 1 5 10 15Glu Ala Leu Ala Gly Glu Lys Gly Phe Gly Ile Asn Pro Val Ile Ile 20 25 30Lys Ser Val Ala Glu Gln Val Ala Glu Val Ala Lys Met Asp Cys Glu 35 40 45Ile Ala Val Ile Val Gly Gly Gly Asn Ile Trp Arg Gly Lys Thr Gly 50 55 60Ser Asp Leu Gly Met Asp Arg Gly Thr Ala Asp Tyr Met Gly Met Leu 65 70 7580 Ala Thr Val Met Asn Ala Leu Ala Leu Gln Asp Ser Leu Glu Gln Leu 85 9095 Asp Cys Asp Thr Arg Val Leu Thr Ser Ile Glu Met Lys Gln Val Ala 100105 110 Glu Pro Tyr Ile Arg Arg Arg Ala Ile Arg His Leu Glu Lys Lys Arg115 120 125 Val Val Ile Phe Ala Ala Gly Ile Gly Asn Pro Tyr Phe Ser ThrAsp 130 135 140 Thr Thr Ala Ala Leu Arg Ala Ala Glu Val Glu Ala Asp ValIle Leu 145 150 155 160 Met Gly Lys Asn Asn Val Asp Gly Val Tyr Ser AlaAsp Pro Lys Val 165 170 175 Asn Lys Asp Ala Val Lys Tyr Glu His Leu ThrHis Ile Gln Met Leu 180 185 190 Gln Glu Gly Leu Gln Val Met Asp Ser ThrAla Ser Ser Phe Cys Met 195 200 205 Asp Asn Asn Ile Pro Leu Thr Val PheSer Ile Met Glu Glu Gly Asn 210 215 220 Ile Lys Arg Ala Val Met Gly GluLys Ile Gly Thr Leu Ile Thr Lys 225 230 235 240 Arg Ser His His His HisHis His 245 89 162 DNA Artificial Sequence DNA sequence of portion ofpQE-60 vector 89 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattataatagattca 60 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaattaaccatggga 120 ggatccagat ctcatcacca tcaccatcac taagcttaat ta 162 90 20DNA Artificial Sequence Oligonucleotide Primer 90 cccgggccat ggctcaaatt20 91 33 DNA Artificial Sequence Oligonucleotide Primer 91 ccatgggattaaagtatgaa catattgcta agc 33 92 40 DNA Artificial SequenceOligonucleotide Primer 92 gagatctctt tttactagga atataaaact gtgcatgacg 4093 31 DNA Artificial Sequence Oligonucleotide Primer 93 gcatgctggcactttatgga tttgcccaag g 31 94 40 DNA Artificial Sequence OligonucleotidePrimer 94 gagatctttt tgattttctt ttaaatcgtt gttcatgatt 40 95 28 DNAArtificial Sequence Oligonucleotide Primer 95 ccatgggatt caaaaacaatttaacatc 28 96 33 DNA Artificial Sequence Oligonucleotide Primer 96gagatctcat gacattttta ataacatttt cgc 33 97 31 DNA Artificial SequenceOligonucleotide Primer 97 ccatgggaat aaataatcat gaattactag g 31 98 36DNA Artificial Sequence Oligonucleotide Primer 98 gagatctacc atgttgacgcttcgtattaa aaggtc 36 99 36 DNA Artificial Sequence OligonucleotidePrimer 99 ccatgggacg tatcttaaaa gagtccatta ttgtgg 36 100 34 DNAArtificial Sequence Oligonucleotide Primer 100 ggatccatcg atatgagatttgccggtaat acgc 34 101 29 DNA Artificial Sequence Oligonucleotide Primer101 ccatgggatt aatcgataca catgtccat 29 102 45 DNA Artificial SequenceOligonucleotide Primer 102 gagatcttga atttaaatta aacaatttct ctgcatttttagttg 45 103 30 DNA Artificial Sequence Oligonucleotide Primer 103ccatgggaat aatatattgg tgtatgacag 30 104 34 DNA Artificial SequenceOligonucleotide Primer 104 gagatctcaa caatacaact ttacctggag ctgg 34 10534 DNA Artificial Sequence Oligonucleotide Primer 105 ccatgggacgatttacattt tcaaacgatt tagg 34 106 36 DNA Artificial SequenceOligonucleotide Primer 106 gagatcttaa aattggcgaa actaattttg ctaacg 36107 35 DNA Artificial Sequence Oligonucleotide Primer 107 ccatgggaaagattttattc gtttgtacag gtaac 35 108 43 DNA Artificial SequenceOligonucleotide Primer 108 gagatctgca aataatatct tttaatttta aaattaaagaatg 43 109 3198 DNA Staphylococcus aureus 109 atggtggcat atttaaatattcatacggct tatgatttgt taaattcaag cttaaaaata 60 gaagatgccg taagacttgctgtgtctgaa aatgttgatg cacttgccat aactgacacc 120 aatgtattgt atggttttcctaaattttat gatgcatgta tagcaaataa cattaaaccg 180 atttttggta tgacaatatatgtgacaaat ggattaaata cagtcgaaac agttgttcta 240 gctaaaaata atgatggattaaaagatttg tatcaactat catcggaaat aaaaatgaat 300 gcattagaac atgtgtcgtttgaattatta aaacgatttt ctaacaatat gattatcatt 360 tttaaaaaag tcggtgatcaacatcgtgat attgtacaag tgtttgaaac ccataatgac 420 acatatatgg accaccttagtatttcgatt caaggtagaa aacatgtttg gattcaaaat 480 gtttgttacc aaacacgtcaagatgccgat acgatttctg cattagcagc tattagagac 540 aatacaaaat tagacttaattcatgatcaa gaagattttg gtgcacattt tttaactgaa 600 aaggaaatta atcaattagatattaaccaa gaatatttaa cgcaggttga tgttatagct 660 caaaagtgtg atgcagaattaaaatatcat caatctctac ttcctcaata tgagacacct 720 aatgatgaat cagctaaaaaatatttgtgg cgtgtcttag ttacacaatt gaaaaaatta 780 gaacttaatt atgacgtctatttagagcga ttgaaatatg agtataaagt tattactaat 840 atgggttttg aagattatttcttaatagta agtgatttaa tccattatgc gaaaacgaat 900 gatgtgatgg taggtcctggtcgtggttct tcagctggct cactggtcag ttatttattg 960 ggaattacaa cgattgatcctattaaattc aatctattat ttgaacgttt tttaaaccca 1020 gaacgtgtaa caatgcctgatattgatatt gactttgaag atacacgccg agaaagggtc 1080 attcagtacg tccaagaaaaatatggcgag ctacatgtat ctggaattgt gactttcggt 1140 catctgcttg caagagcagttgctagagat gttggaagaa ttatggggtt tgatgaagtt 1200 acattaaatg aaatttcaagtttaatccca cataaattag gaattacact tgatgaagca 1260 tatcaaattg acgattttaaagagtttgta catcgaaacc atcgacatga acgctggttc 1320 agtatttgta aaaagttagaaggtttacca agacatacat ctacacatgc ggcaggaatt 1380 attattaatg accatccattatatgaatat gcccctttaa cgaaagggga tacaggatta 1440 ttaacgcaat ggacaatgactgaagccgaa cgtattgggt tattaaaaat agattttcta 1500 gggttgagaa acttatcgattattcatcaa atcttaacac aagtcaaaaa agatttaggt 1560 attaatattg atatcgaaaagattccgttt gatgatcaaa aagtgtttga attgttgtcg 1620 caaggagata cgactggcatattccaatta gagtctgacg gtgtaagaag tgtattaaaa 1680 aaattaaagc cggaacactttgaagatatt gttgctgtaa cttctttgta tagaccaggt 1740 ccaatggaag aaattccaacttacattaca agaagacatg atccaagcaa agttcaatat 1800 ttacatccgc atttagaacctatattaaaa aatacttacg gtgttattat ttatcaagag 1860 caaattatgc aaatagcgagcacatttgca aacttcagtt atggtgaagc ggatatttta 1920 agaagagcaa tgagtaaaaaaaatagagct gttcttgaaa gtgagcgtca acattttata 1980 gaaggtgcaa agcaaaatggttatcacgaa gacattagta agcaaatatt tgatttgatt 2040 ctgaaatttg ctgattatggttttcctaga gcacatgctg tcagctattc taaaattgca 2100 tacattatga gctttttaaaagtccattat ccaaattatt tttacgcaaa tattttaagt 2160 aatgttattg gaagtgagaagaaaactgct caaatgatag aagaagcaaa aaaacaaggt 2220 atcactatat tgccaccgaacattaacgaa agtcattggt tttataaacc ttcccaagaa 2280 ggcatttatt tatcaattggtacaattaaa ggtgttggtt atcaaagtgt gaaagtgatt 2340 gttgatgaac gttatcagaacggcaaattt aaagatttct ttgattttgc tagacgtata 2400 ccgaagagag tcaaaacgagaaagttactt gaagcactga ttttagtggg agcgtttgat 2460 gcttttggta aaacacgttcaacgttgttg caagctattg atcaagtgtt ggatggcgat 2520 tttaaaacat tggaacaagatggtttttta tttgatattt taacgccaaa acagatgtat 2580 gaagataaag aagaattgcctgatgcactt attagtcagt acgaaaaaga atatttagga 2640 ttttatgttt cgcaacacccagtagataaa aagtttgttg ccaaacaata tttaacgata 2700 tttaaattga gtaacgcgcagaattataaa cctatattag tacagtttga taaagttaaa 2760 caaattcgaa ctaaaaatggtcaaaatatg gcattcgtca cattaaatga tggcattgaa 2820 actttagatg gtgtgattttccctaatcag tttaaaaagt acgaagagtt gttatcacat 2880 aatgacttgt ttatagttagcgggaaattt gaccatagaa agcaacaacg tcaactaatt 2940 ataaatgaga ttcagacattagccactttt gaagaacaaa aattagcatt tgccaaacaa 3000 attataatta gaaataaatcacaaatagat atgtttgaag agatgattaa agctacgaaa 3060 gagaatgcta atgatgttgtgttatccttt tatgatgaaa cgattaaaca aatgactact 3120 ttaggctata ttaatcaaaaagatagtatg tttaataatt ttatacaatc ctttaaccct 3180 agtgatatta ggcttata3198 110 1066 PRT Staphylococcus aureus 110 Met Val Ala Tyr Leu Asn IleHis Thr Ala Tyr Asp Leu Leu Asn Ser 1 5 10 15 Ser Leu Lys Ile Glu AspAla Val Arg Leu Ala Val Ser Glu Asn Val 20 25 30 Asp Ala Leu Ala Ile ThrAsp Thr Asn Val Leu Tyr Gly Phe Pro Lys 35 40 45 Phe Tyr Asp Ala Cys IleAla Asn Asn Ile Lys Pro Ile Phe Gly Met 50 55 60 Thr Ile Tyr Val Thr AsnGly Leu Asn Thr Val Glu Thr Val Val Leu 65 70 75 80 Ala Lys Asn Asn AspGly Leu Lys Asp Leu Tyr Gln Leu Ser Ser Glu 85 90 95 Ile Lys Met Asn AlaLeu Glu His Val Ser Phe Glu Leu Leu Lys Arg 100 105 110 Phe Ser Asn AsnMet Ile Ile Ile Phe Lys Lys Val Gly Asp Gln His 115 120 125 Arg Asp IleVal Gln Val Phe Glu Thr His Asn Asp Thr Tyr Met Asp 130 135 140 His LeuSer Ile Ser Ile Gln Gly Arg Lys His Val Trp Ile Gln Asn 145 150 155 160Val Cys Tyr Gln Thr Arg Gln Asp Ala Asp Thr Ile Ser Ala Leu Ala 165 170175 Ala Ile Arg Asp Asn Thr Lys Leu Asp Leu Ile His Asp Gln Glu Asp 180185 190 Phe Gly Ala His Phe Leu Thr Glu Lys Glu Ile Asn Gln Leu Asp Ile195 200 205 Asn Gln Glu Tyr Leu Thr Gln Val Asp Val Ile Ala Gln Lys CysAsp 210 215 220 Ala Glu Leu Lys Tyr His Gln Ser Leu Leu Pro Gln Tyr GluThr Pro 225 230 235 240 Asn Asp Glu Ser Ala Lys Lys Tyr Leu Trp Arg ValLeu Val Thr Gln 245 250 255 Leu Lys Lys Leu Glu Leu Asn Tyr Asp Val TyrLeu Glu Arg Leu Lys 260 265 270 Tyr Glu Tyr Lys Val Ile Thr Asn Met GlyPhe Glu Asp Tyr Phe Leu 275 280 285 Ile Val Ser Asp Leu Ile His Tyr AlaLys Thr Asn Asp Val Met Val 290 295 300 Gly Pro Gly Arg Gly Ser Ser AlaGly Ser Leu Val Ser Tyr Leu Leu 305 310 315 320 Gly Ile Thr Thr Ile AspPro Ile Lys Phe Asn Leu Leu Phe Glu Arg 325 330 335 Phe Leu Asn Pro GluArg Val Thr Met Pro Asp Ile Asp Ile Asp Phe 340 345 350 Glu Asp Thr ArgArg Glu Arg Val Ile Gln Tyr Val Gln Glu Lys Tyr 355 360 365 Gly Glu LeuHis Val Ser Gly Ile Val Thr Phe Gly His Leu Leu Ala 370 375 380 Arg AlaVal Ala Arg Asp Val Gly Arg Ile Met Gly Phe Asp Glu Val 385 390 395 400Thr Leu Asn Glu Ile Ser Ser Leu Ile Pro His Lys Leu Gly Ile Thr 405 410415 Leu Asp Glu Ala Tyr Gln Ile Asp Asp Phe Lys Glu Phe Val His Arg 420425 430 Asn His Arg His Glu Arg Trp Phe Ser Ile Cys Lys Lys Leu Glu Gly435 440 445 Leu Pro Arg His Thr Ser Thr His Ala Ala Gly Ile Ile Ile AsnAsp 450 455 460 His Pro Leu Tyr Glu Tyr Ala Pro Leu Thr Lys Gly Asp ThrGly Leu 465 470 475 480 Leu Thr Gln Trp Thr Met Thr Glu Ala Glu Arg IleGly Leu Leu Lys 485 490 495 Ile Asp Phe Leu Gly Leu Arg Asn Leu Ser IleIle His Gln Ile Leu 500 505 510 Thr Gln Val Lys Lys Asp Leu Gly Ile AsnIle Asp Ile Glu Lys Ile 515 520 525 Pro Phe Asp Asp Gln Lys Val Phe GluLeu Leu Ser Gln Gly Asp Thr 530 535 540 Thr Gly Ile Phe Gln Leu Glu SerAsp Gly Val Arg Ser Val Leu Lys 545 550 555 560 Lys Leu Lys Pro Glu HisPhe Glu Asp Ile Val Ala Val Thr Ser Leu 565 570 575 Tyr Arg Pro Gly ProMet Glu Glu Ile Pro Thr Tyr Ile Thr Arg Arg 580 585 590 His Asp Pro SerLys Val Gln Tyr Leu His Pro His Leu Glu Pro Ile 595 600 605 Leu Lys AsnThr Tyr Gly Val Ile Ile Tyr Gln Glu Gln Ile Met Gln 610 615 620 Ile AlaSer Thr Phe Ala Asn Phe Ser Tyr Gly Glu Ala Asp Ile Leu 625 630 635 640Arg Arg Ala Met Ser Lys Lys Asn Arg Ala Val Leu Glu Ser Glu Arg 645 650655 Gln His Phe Ile Glu Gly Ala Lys Gln Asn Gly Tyr His Glu Asp Ile 660665 670 Ser Lys Gln Ile Phe Asp Leu Ile Leu Lys Phe Ala Asp Tyr Gly Phe675 680 685 Pro Arg Ala His Ala Val Ser Tyr Ser Lys Ile Ala Tyr Ile MetSer 690 695 700 Phe Leu Lys Val His Tyr Pro Asn Tyr Phe Tyr Ala Asn IleLeu Ser 705 710 715 720 Asn Val Ile Gly Ser Glu Lys Lys Thr Ala Gln MetIle Glu Glu Ala 725 730 735 Lys Lys Gln Gly Ile Thr Ile Leu Pro Pro AsnIle Asn Glu Ser His 740 745 750 Trp Phe Tyr Lys Pro Ser Gln Glu Gly IleTyr Leu Ser Ile Gly Thr 755 760 765 Ile Lys Gly Val Gly Tyr Gln Ser ValLys Val Ile Val Asp Glu Arg 770 775 780 Tyr Gln Asn Gly Lys Phe Lys AspPhe Phe Asp Phe Ala Arg Arg Ile 785 790 795 800 Pro Lys Arg Val Lys ThrArg Lys Leu Leu Glu Ala Leu Ile Leu Val 805 810 815 Gly Ala Phe Asp AlaPhe Gly Lys Thr Arg Ser Thr Leu Leu Gln Ala 820 825 830 Ile Asp Gln ValLeu Asp Gly Asp Phe Lys Thr Leu Glu Gln Asp Gly 835 840 845 Phe Leu PheAsp Ile Leu Thr Pro Lys Gln Met Tyr Glu Asp Lys Glu 850 855 860 Glu LeuPro Asp Ala Leu Ile Ser Gln Tyr Glu Lys Glu Tyr Leu Gly 865 870 875 880Phe Tyr Val Ser Gln His Pro Val Asp Lys Lys Phe Val Ala Lys Gln 885 890895 Tyr Leu Thr Ile Phe Lys Leu Ser Asn Ala Gln Asn Tyr Lys Pro Ile 900905 910 Leu Val Gln Phe Asp Lys Val Lys Gln Ile Arg Thr Lys Asn Gly Gln915 920 925 Asn Met Ala Phe Val Thr Leu Asn Asp Gly Ile Glu Thr Leu AspGly 930 935 940 Val Ile Phe Pro Asn Gln Phe Lys Lys Tyr Glu Glu Leu LeuSer His 945 950 955 960 Asn Asp Leu Phe Ile Val Ser Gly Lys Phe Asp HisArg Lys Gln Gln 965 970 975 Arg Gln Leu Ile Ile Asn Glu Ile Gln Thr LeuAla Thr Phe Glu Glu 980 985 990 Gln Lys Leu Ala Phe Ala Lys Gln Ile IleIle Arg Asn Lys Ser Gln 995 1000 1005 Ile Asp Met Phe Glu Glu Met IleLys Ala Thr Lys Glu Asn Ala 1010 1015 1020 Asn Asp Val Val Leu Ser PheTyr Asp Glu Thr Ile Lys Gln Met 1025 1030 1035 Thr Thr Leu Gly Tyr IleAsn Gln Lys Asp Ser Met Phe Asn Asn 1040 1045 1050 Phe Ile Gln Ser PheAsn Pro Ser Asp Ile Arg Leu Ile 1055 1060 1065 111 21 DNA ArtificialSequence Oligonucleotide Primer 111 tagaagatgc cgtaagactt g 21 112 21DNA Artificial Sequence Oligonucleotide Primer 112 atatcacgat gttgatcaccg 21 113 825 DNA Staphylococcus aureus 113 atgaagaaaa aagcgttactaccattattt ttaggtatta tggtcttttt ggctggttgt 60 gactattcta aacctgaaaaacgtagtggg tttttctaca atacattcgt agatccaatg 120 aaaaatgtat tggattggttgggaaataac ttattaaacg acaattatgg tttagctatt 180 attatccttg tattggtaattcgtattatt ttattaccat tcatgttgtc aaactataaa 240 aatagtcata tgatgcgtcaaaaaatgaaa gttgcaaagc cagaagttga aaaaattcaa 300 gaaaaagtga aacgtgcgcgtacacaagaa gaaaaaatgg ctgcaaacca agaattaatg 360 caagtatata aaaagtatgacatgaacccg attaagagta tgttgggttg tttaccaatg 420 ctaatccaat taccaatcatcatgggatta tactttgtac ttaaagacca acttgtagat 480 ggtttgttta aatatccacacttcttatgg ttcgatttag gacgtcctga tatttggatt 540 acaattattg ccggtgttttatactttatc caagcatatg tatcaagtaa aacgatgcca 600 gacgaacaac gtcaaatgggttacatgatg atggtcattt caccaattat gattatctgg 660 atttcattaa gctcagcatcagcacttggt ttgtactggt cagtcagtgc ggcgttcctt 720 gtagttcaaa cacactttgcgaacatttat tatgaaaaag tcgctaaaaa agaagtacaa 780 cctttcattg aagcgtatgaaagagagcac aacggcggca gcaat 825 114 275 PRT Staphylococcus aureus 114Met Lys Lys Lys Ala Leu Leu Pro Leu Phe Leu Gly Ile Met Val Phe 1 5 1015 Leu Ala Gly Cys Asp Tyr Ser Lys Pro Glu Lys Arg Ser Gly Phe Phe 20 2530 Tyr Asn Thr Phe Val Asp Pro Met Lys Asn Val Leu Asp Trp Leu Gly 35 4045 Asn Asn Leu Leu Asn Asp Asn Tyr Gly Leu Ala Ile Ile Ile Leu Val 50 5560 Leu Val Ile Arg Ile Ile Leu Leu Pro Phe Met Leu Ser Asn Tyr Lys 65 7075 80 Asn Ser His Met Met Arg Gln Lys Met Lys Val Ala Lys Pro Glu Val 8590 95 Glu Lys Ile Gln Glu Lys Val Lys Arg Ala Arg Thr Gln Glu Glu Lys100 105 110 Met Ala Ala Asn Gln Glu Leu Met Gln Val Tyr Lys Lys Tyr AspMet 115 120 125 Asn Pro Ile Lys Ser Met Leu Gly Cys Leu Pro Met Leu IleGln Leu 130 135 140 Pro Ile Ile Met Gly Leu Tyr Phe Val Leu Lys Asp GlnLeu Val Asp 145 150 155 160 Gly Leu Phe Lys Tyr Pro His Phe Leu Trp PheAsp Leu Gly Arg Pro 165 170 175 Asp Ile Trp Ile Thr Ile Ile Ala Gly ValLeu Tyr Phe Ile Gln Ala 180 185 190 Tyr Val Ser Ser Lys Thr Met Pro AspGlu Gln Arg Gln Met Gly Tyr 195 200 205 Met Met Met Val Ile Ser Pro IleMet Ile Ile Trp Ile Ser Leu Ser 210 215 220 Ser Ala Ser Ala Leu Gly LeuTyr Trp Ser Val Ser Ala Ala Phe Leu 225 230 235 240 Val Val Gln Thr HisPhe Ala Asn Ile Tyr Tyr Glu Lys Val Ala Lys 245 250 255 Lys Glu Val GlnPro Phe Ile Glu Ala Tyr Glu Arg Glu His Asn Gly 260 265 270 Gly Ser Asn275 115 21 DNA Artificial Sequence Oligonucleotide Primer 115 tatggtctttttggctggtt g 21 116 21 DNA Artificial Sequence Oligonucleotide Primer116 tttttcttct tgtgtacgcg c 21 117 807 DNA Staphylococcus aureus 117atggattttt ccaacttttt tcaaaacctc agtacgttaa aaattgtaac gagtatcctt 60gatttactga tagtttggta tgtactttat cttctcatca cggtctttaa gggaactaaa 120gcgatacaat tacttaaagg gatattagta attgttattg gtcagcagat aagtatgata 180ttgaacttga ctgcaacatc taaattattc gatatcgtta ttcaatgggg ggtattagct 240ttaatagtaa tattccaacc agaaattaga cgtgcgttag aacaacttgg tagaggtagc 300tttttaaaac gctatacttc taatacgtat agtaaagatg aagagaaatt gattcaatcg 360gtttcaaagg ctgtgcaata tatggctaaa agacgtatag gtgcattaat tgtctttgaa 420aaagaaacag gtcttcaaga ttatattgaa acaggtattg caatggattc aaatatttcg 480caagaacttt taattaatgt ctttatacct aacacacctt tacatgatgg tgcaatgatt 540attcaaggca cgaagattgc agcagcagca agttatttgc cattgtctga tagtcctaag 600atatctaaaa gtttgggtac aagacataga gctgcggttg gtatttcaga agtatctgat 660gcatttaccg ttattgtatc tgaagaaact ggtgatattt cggtaacatt tgatggaaaa 720ttacgacgag acatttcaaa cgaaattttt gaagagttgc ttgctgaaca ttggtttggc 780acacgctttc aaaagaaagg tgtgaaa 807 118 269 PRT Staphylococcus aureus 118Met Asp Phe Ser Asn Phe Phe Gln Asn Leu Ser Thr Leu Lys Ile Val 1 5 1015 Thr Ser Ile Leu Asp Leu Leu Ile Val Trp Tyr Val Leu Tyr Leu Leu 20 2530 Ile Thr Val Phe Lys Gly Thr Lys Ala Ile Gln Leu Leu Lys Gly Ile 35 4045 Leu Val Ile Val Ile Gly Gln Gln Ile Ser Met Ile Leu Asn Leu Thr 50 5560 Ala Thr Ser Lys Leu Phe Asp Ile Val Ile Gln Trp Gly Val Leu Ala 65 7075 80 Leu Ile Val Ile Phe Gln Pro Glu Ile Arg Arg Ala Leu Glu Gln Leu 8590 95 Gly Arg Gly Ser Phe Leu Lys Arg Tyr Thr Ser Asn Thr Tyr Ser Lys100 105 110 Asp Glu Glu Lys Leu Ile Gln Ser Val Ser Lys Ala Val Gln TyrMet 115 120 125 Ala Lys Arg Arg Ile Gly Ala Leu Ile Val Phe Glu Lys GluThr Gly 130 135 140 Leu Gln Asp Tyr Ile Glu Thr Gly Ile Ala Met Asp SerAsn Ile Ser 145 150 155 160 Gln Glu Leu Leu Ile Asn Val Phe Ile Pro AsnThr Pro Leu His Asp 165 170 175 Gly Ala Met Ile Ile Gln Gly Thr Lys IleAla Ala Ala Ala Ser Tyr 180 185 190 Leu Pro Leu Ser Asp Ser Pro Lys IleSer Lys Ser Leu Gly Thr Arg 195 200 205 His Arg Ala Ala Val Gly Ile SerGlu Val Ser Asp Ala Phe Thr Val 210 215 220 Ile Val Ser Glu Glu Thr GlyAsp Ile Ser Val Thr Phe Asp Gly Lys 225 230 235 240 Leu Arg Arg Asp IleSer Asn Glu Ile Phe Glu Glu Leu Leu Ala Glu 245 250 255 His Trp Phe GlyThr Arg Phe Gln Lys Lys Gly Val Lys 260 265 119 21 DNA ArtificialSequence Oligonucleotide Primer 119 ctttatcttc tcatcacggt c 21 120 21DNA Artificial Sequence Oligonucleotide Primer 120 tgcacctata cgtcttttagc 21 121 606 DNA Staphylococcus aureus 121 atgatgataa tcgtcatgttactactaagt tatcttatcg gcgctttccc aagtggattc 60 gtaattggaa aattatttttcaaaaaagat attagacaat ttggtagtgg taatactggc 120 gctactaata gctttagagtattaggtcgt cctgcaggat tcttggtaac atttctagat 180 attttcaaag ggttcataactgttttcttc cctttatggt tacaagttca cgcagatggc 240 cctattagta ctttttttacaaatggttta attgttggct tattcgctat acttggacac 300 gtttatcctg tttatttaaaattccaaggt ggcaaagctg ttgcaactag tgcaggtgtc 360 gtcttgggag tcaatccgatacttttacta atacttgcaa ttatcttctt tattgtattg 420 aagattttta aatatgtttctttagcaagt atcgttgcag caatttgctg tgtgattggc 480 tcgcttatca ttcaagactatattttatta gtcgttagtt tcttagtttc aatcatattg 540 ataattagac atcgctctaatatcgcaagg atttttagag gcgaagaacc taaaataaaa 600 tggatg 606 122 202 PRTStaphylococcus aureus 122 Met Met Ile Ile Val Met Leu Leu Leu Ser TyrLeu Ile Gly Ala Phe 1 5 10 15 Pro Ser Gly Phe Val Ile Gly Lys Leu PhePhe Lys Lys Asp Ile Arg 20 25 30 Gln Phe Gly Ser Gly Asn Thr Gly Ala ThrAsn Ser Phe Arg Val Leu 35 40 45 Gly Arg Pro Ala Gly Phe Leu Val Thr PheLeu Asp Ile Phe Lys Gly 50 55 60 Phe Ile Thr Val Phe Phe Pro Leu Trp LeuGln Val His Ala Asp Gly 65 70 75 80 Pro Ile Ser Thr Phe Phe Thr Asn GlyLeu Ile Val Gly Leu Phe Ala 85 90 95 Ile Leu Gly His Val Tyr Pro Val TyrLeu Lys Phe Gln Gly Gly Lys 100 105 110 Ala Val Ala Thr Ser Ala Gly ValVal Leu Gly Val Asn Pro Ile Leu 115 120 125 Leu Leu Ile Leu Ala Ile IlePhe Phe Ile Val Leu Lys Ile Phe Lys 130 135 140 Tyr Val Ser Leu Ala SerIle Val Ala Ala Ile Cys Cys Val Ile Gly 145 150 155 160 Ser Leu Ile IleGln Asp Tyr Ile Leu Leu Val Val Ser Phe Leu Val 165 170 175 Ser Ile IleLeu Ile Ile Arg His Arg Ser Asn Ile Ala Arg Ile Phe 180 185 190 Arg GlyGlu Glu Pro Lys Ile Lys Trp Met 195 200 123 21 DNA Artificial SequenceOligonucleotide Primer 123 gttatcttat cggcgctttc c 21 124 21 DNAArtificial Sequence Oligonucleotide Primer 124 gcactagttg caacagcttt g21 125 1293 DNA Staphylococcus aureus 125 atgcagttaa atagtaatggttggcatgtt gatgaccata ttgttgtcgc tgtttctaca 60 ggtattgata gtatgtgtttattgtatcaa ctactaaatg attataaaga tagttataga 120 aaactaacat gcttacatgtcaatcatggc gttaggtcag cttcaattga ggaagccaga 180 tttttagaag catactgcgaacgtcatcac atcgatttac atatcaaaaa gttagatttg 240 tcgcatagtc tcgaccgaaataacagcatt cagaatgaag ctcgaattaa acgttacgaa 300 tggtttgatg aaatgatgaatgtattagaa gcggatgtat tgctaacggc gcatcatttg 360 gacgatcaat tagaaactattatgtatcgt atttttaatg ggaaatcaac acgtaataaa 420 ctaggatttg atgagttatcgaagcgaaaa ggttatcaga tttatcgacc acttttagct 480 gtctctaaaa aagaaataaaacaattccaa gagagatatc atattccata ttttgaagat 540 gaatctaata aagataacaaatatgttaga aatgatattc gtaatagaat tattccagct 600 attgatgaaa ataatcaacttaaagtatcg catttattaa aattaaaaca atggcatgat 660 gaacaatatg atattttgcaatattcagct aaacaattta ttcaagaatt tgtgaagttt 720 gatgaacagt caaaatatttagaggtttct agacaagctt ttaataactt accaaactca 780 ttaaagatgg ttgtgttggactgcctatta tcaaagtatt atgagttgtt taatattagt 840 gctaaaacat acgaagagtggtttaaacaa tttagtagta agaaagcaca attcagtatt 900 aatctcacgg ataaatggataattcaaatc gcatatggta aattaataat aatggctaaa 960 aataatggcg atacatattttagagttcaa acaattaaaa agccaggtaa ttatattttt 1020 aacaaatatc gattagagatacattctaat ttaccaaaat gtttatttcc gcttacagtg 1080 agaacacgac aaagtggcgatacatttaaa ctgaatgggc gcgatggtta taagaaagtg 1140 aatcgcctgt ttatagattgtaaagtgcca cagtgggttc gggatcaaat gccaatcgta 1200 ttggataaac aacagcgcattattgcggta ggagatttat atcaacaaca aacaataaaa 1260 aaatggatta taattagtaaaaatggagat gaa 1293 126 431 PRT Staphylococcus aureus 126 Met Gln LeuAsn Ser Asn Gly Trp His Val Asp Asp His Ile Val Val 1 5 10 15 Ala ValSer Thr Gly Ile Asp Ser Met Cys Leu Leu Tyr Gln Leu Leu 20 25 30 Asn AspTyr Lys Asp Ser Tyr Arg Lys Leu Thr Cys Leu His Val Asn 35 40 45 His GlyVal Arg Ser Ala Ser Ile Glu Glu Ala Arg Phe Leu Glu Ala 50 55 60 Tyr CysGlu Arg His His Ile Asp Leu His Ile Lys Lys Leu Asp Leu 65 70 75 80 SerHis Ser Leu Asp Arg Asn Asn Ser Ile Gln Asn Glu Ala Arg Ile 85 90 95 LysArg Tyr Glu Trp Phe Asp Glu Met Met Asn Val Leu Glu Ala Asp 100 105 110Val Leu Leu Thr Ala His His Leu Asp Asp Gln Leu Glu Thr Ile Met 115 120125 Tyr Arg Ile Phe Asn Gly Lys Ser Thr Arg Asn Lys Leu Gly Phe Asp 130135 140 Glu Leu Ser Lys Arg Lys Gly Tyr Gln Ile Tyr Arg Pro Leu Leu Ala145 150 155 160 Val Ser Lys Lys Glu Ile Lys Gln Phe Gln Glu Arg Tyr HisIle Pro 165 170 175 Tyr Phe Glu Asp Glu Ser Asn Lys Asp Asn Lys Tyr ValArg Asn Asp 180 185 190 Ile Arg Asn Arg Ile Ile Pro Ala Ile Asp Glu AsnAsn Gln Leu Lys 195 200 205 Val Ser His Leu Leu Lys Leu Lys Gln Trp HisAsp Glu Gln Tyr Asp 210 215 220 Ile Leu Gln Tyr Ser Ala Lys Gln Phe IleGln Glu Phe Val Lys Phe 225 230 235 240 Asp Glu Gln Ser Lys Tyr Leu GluVal Ser Arg Gln Ala Phe Asn Asn 245 250 255 Leu Pro Asn Ser Leu Lys MetVal Val Leu Asp Cys Leu Leu Ser Lys 260 265 270 Tyr Tyr Glu Leu Phe AsnIle Ser Ala Lys Thr Tyr Glu Glu Trp Phe 275 280 285 Lys Gln Phe Ser SerLys Lys Ala Gln Phe Ser Ile Asn Leu Thr Asp 290 295 300 Lys Trp Ile IleGln Ile Ala Tyr Gly Lys Leu Ile Ile Met Ala Lys 305 310 315 320 Asn AsnGly Asp Thr Tyr Phe Arg Val Gln Thr Ile Lys Lys Pro Gly 325 330 335 AsnTyr Ile Phe Asn Lys Tyr Arg Leu Glu Ile His Ser Asn Leu Pro 340 345 350Lys Cys Leu Phe Pro Leu Thr Val Arg Thr Arg Gln Ser Gly Asp Thr 355 360365 Phe Lys Leu Asn Gly Arg Asp Gly Tyr Lys Lys Val Asn Arg Leu Phe 370375 380 Ile Asp Cys Lys Val Pro Gln Trp Val Arg Asp Gln Met Pro Ile Val385 390 395 400 Leu Asp Lys Gln Gln Arg Ile Ile Ala Val Gly Asp Leu TyrGln Gln 405 410 415 Gln Thr Ile Lys Lys Trp Ile Ile Ile Ser Lys Asn GlyAsp Glu 420 425 430 127 21 DNA Artificial Sequence OligonucleotidePrimer 127 aatggttggc atgttgatga c 21 128 21 DNA Artificial SequenceOligonucleotide Primer 128 attgatcgtc caaatgatgc g 21 129 429 DNAStaphylococcus aureus 129 atgttcatgg gagaatacga tcatcaatta gatacaaaaggacgtatgat tataccgtcc 60 aagtttcgtt atgacttaaa tgagcgtttt attatcacaagaggccttga taaatgttta 120 ttcggttaca ctctagacga atggcaacag attgaagagaaaatgaaaac cttacctatg 180 acaaaaaaag acgcacgtaa gtttatgcgt atgttcttctctggtgctgt tgaagtagaa 240 cttgataagc aagggcgtat taacatccct caaaacttgaggaaatacgc taatttaact 300 aaagaatgta cagtaatcgg tgtttcaaat cgtattgagatttgggatag agaaacttgg 360 aatgatttct atgaagaatc tgaagaaagt ttcgaagatattgctgaaga tttaatagat 420 tttgatttt 429 130 143 PRT Staphylococcusaureus 130 Met Phe Met Gly Glu Tyr Asp His Gln Leu Asp Thr Lys Gly ArgMet 1 5 10 15 Ile Ile Pro Ser Lys Phe Arg Tyr Asp Leu Asn Glu Arg PheIle Ile 20 25 30 Thr Arg Gly Leu Asp Lys Cys Leu Phe Gly Tyr Thr Leu AspGlu Trp 35 40 45 Gln Gln Ile Glu Glu Lys Met Lys Thr Leu Pro Met Thr LysLys Asp 50 55 60 Ala Arg Lys Phe Met Arg Met Phe Phe Ser Gly Ala Val GluVal Glu 65 70 75 80 Leu Asp Lys Gln Gly Arg Ile Asn Ile Pro Gln Asn LeuArg Lys Tyr 85 90 95 Ala Asn Leu Thr Lys Glu Cys Thr Val Ile Gly Val SerAsn Arg Ile 100 105 110 Glu Ile Trp Asp Arg Glu Thr Trp Asn Asp Phe TyrGlu Glu Ser Glu 115 120 125 Glu Ser Phe Glu Asp Ile Ala Glu Asp Leu IleAsp Phe Asp Phe 130 135 140 131 22 DNA Artificial SequenceOligonucleotide Primer 131 ttcatgggag aatacgatca tc 22 132 22 DNAArtificial Sequence Oligonucleotide Primer 132 gtatttcctc aagttttgag gg22 133 741 DNA Staphylococcus aureus 133 atgctagagg cacaattttttactgatact ggacaacata gagataagaa tgaagatgcg 60 ggtggtattt tttataatcaaactaatcaa caacttttag ttctgtgtga tggtatgggt 120 ggccataaag caggagaagttgcaagtaaa tttgttacag atgagttgaa atcccgtttt 180 gaagcggaaa atcttatagaacaacatcaa gctgaaaatt ggttgcgtaa taatataaaa 240 gatataaatt ttcagttatatcactatgca caagaaaatg cagaatataa aggtatgggt 300 acaacatgtg tttgtgcacttgtttttgaa aaatcagttg tgatagcaaa tgtcggtgat 360 tctagagcct atgttattaatagtcgacaa attgaacaaa ttactagtga tcactcattt 420 gttaatcatc ttgttttaacgggtcaaatt acgccggaag aagcatttac acatccacaa 480 cgtaatatta ttacgaaggtgatgggcaca gataaacgtg tgagtccaga tttgtttatt 540 aagcgattaa atttttatgattatttatta ttaaattcag atggattaac tgattatgtt 600 aaagacaatg aaattaagcgtttgttagta aaagaaggta caatagaaga tcatggtgat 660 caattaatgc aattggcattagataaccat tcgaaagata acgttacttt catactcgcg 720 gctattgaag gtgataaagt a741 134 247 PRT Staphylococcus aureus 134 Met Leu Glu Ala Gln Phe PheThr Asp Thr Gly Gln His Arg Asp Lys 1 5 10 15 Asn Glu Asp Ala Gly GlyIle Phe Tyr Asn Gln Thr Asn Gln Gln Leu 20 25 30 Leu Val Leu Cys Asp GlyMet Gly Gly His Lys Ala Gly Glu Val Ala 35 40 45 Ser Lys Phe Val Thr AspGlu Leu Lys Ser Arg Phe Glu Ala Glu Asn 50 55 60 Leu Ile Glu Gln His GlnAla Glu Asn Trp Leu Arg Asn Asn Ile Lys 65 70 75 80 Asp Ile Asn Phe GlnLeu Tyr His Tyr Ala Gln Glu Asn Ala Glu Tyr 85 90 95 Lys Gly Met Gly ThrThr Cys Val Cys Ala Leu Val Phe Glu Lys Ser 100 105 110 Val Val Ile AlaAsn Val Gly Asp Ser Arg Ala Tyr Val Ile Asn Ser 115 120 125 Arg Gln IleGlu Gln Ile Thr Ser Asp His Ser Phe Val Asn His Leu 130 135 140 Val LeuThr Gly Gln Ile Thr Pro Glu Glu Ala Phe Thr His Pro Gln 145 150 155 160Arg Asn Ile Ile Thr Lys Val Met Gly Thr Asp Lys Arg Val Ser Pro 165 170175 Asp Leu Phe Ile Lys Arg Leu Asn Phe Tyr Asp Tyr Leu Leu Leu Asn 180185 190 Ser Asp Gly Leu Thr Asp Tyr Val Lys Asp Asn Glu Ile Lys Arg Leu195 200 205 Leu Val Lys Glu Gly Thr Ile Glu Asp His Gly Asp Gln Leu MetGln 210 215 220 Leu Ala Leu Asp Asn His Ser Lys Asp Asn Val Thr Phe IleLeu Ala 225 230 235 240 Ala Ile Glu Gly Asp Lys Val 245 135 21 DNAArtificial Sequence Oligonucleotide Primer 135 ctgatactgg acaacataga g21 136 21 DNA Artificial Sequence Oligonucleotide Primer 136 accgacatttgctatcacaa c 21 137 567 DNA Staphylococcus aureus 137 atgaaaaagatagtacttta cggcggtcag tttaacccta tccatactgc acatatgata 60 gtagctagcgaagtatttca tgaattacag ccagatgaat tttatttttt acctagtttt 120 atgtctccattgaaaaagca ccatgatttt atagacgttc agcacagatt aacaatgata 180 cagatgattatcgacgagct tggttttgga gatatttgtg acgatgaaat taaacgtggt 240 ggtcaaagttatacctatga cacgatcaag gcattcaagg agcaacacaa agacagtgag 300 ttgtactttgttattgggac ggatcagtat aaccaactag agaaatggta tcaaattgaa 360 tacttaaaagaaatggttac ttttgtagtt gtaaatcgag acaaaaatag tcaaaatgtt 420 gaaaatgctatgattgcaat tcagatacct agggtagata taagttcgac aatgattcga 480 caaagagttagtgaagggaa atctatccaa gttcttgttc ctaaatccgt tgaaaactat 540 attaagggggaaggattata tgaacat 567 138 189 PRT Staphylococcus aureus 138 Met Lys LysIle Val Leu Tyr Gly Gly Gln Phe Asn Pro Ile His Thr 1 5 10 15 Ala HisMet Ile Val Ala Ser Glu Val Phe His Glu Leu Gln Pro Asp 20 25 30 Glu PheTyr Phe Leu Pro Ser Phe Met Ser Pro Leu Lys Lys His His 35 40 45 Asp PheIle Asp Val Gln His Arg Leu Thr Met Ile Gln Met Ile Ile 50 55 60 Asp GluLeu Gly Phe Gly Asp Ile Cys Asp Asp Glu Ile Lys Arg Gly 65 70 75 80 GlyGln Ser Tyr Thr Tyr Asp Thr Ile Lys Ala Phe Lys Glu Gln His 85 90 95 LysAsp Ser Glu Leu Tyr Phe Val Ile Gly Thr Asp Gln Tyr Asn Gln 100 105 110Leu Glu Lys Trp Tyr Gln Ile Glu Tyr Leu Lys Glu Met Val Thr Phe 115 120125 Val Val Val Asn Arg Asp Lys Asn Ser Gln Asn Val Glu Asn Ala Met 130135 140 Ile Ala Ile Gln Ile Pro Arg Val Asp Ile Ser Ser Thr Met Ile Arg145 150 155 160 Gln Arg Val Ser Glu Gly Lys Ser Ile Gln Val Leu Val ProLys Ser 165 170 175 Val Glu Asn Tyr Ile Lys Gly Glu Gly Leu Tyr Glu His180 185 139 21 DNA Artificial Sequence Oligonucleotide Primer 139gtttaaccct atccatactg c 21 140 21 DNA Artificial SequenceOligonucleotide Primer 140 ctagttggtt atactgatcc g 21 141 393 DNAStaphylococcus aureus 141 atggtaacat tatttacttc accaagttgc acatcttgccgtaaagcgaa agcatggtta 60 caagaacatg acattccgta tacggagcgt aatattttttctgaacattt aacaattgat 120 gaaattaagc aaatattaaa aatgactgaa gacggtactgatgaaatcat ttctacacgt 180 tctaaaacat accaaaaatt aaatgttgat attgattcactaccattaca agacttatat 240 tcaatcattc aagataatcc tggcttatta cgtcgtccaattattttaga taataaacga 300 ctacaagttg gttataatga ggacgagatt cgacgtttcttacctagaaa agttcgtacg 360 ttccaattac aagaagcaca acgtatggtt gac 393 142131 PRT Staphylococcus aureus 142 Met Val Thr Leu Phe Thr Ser Pro SerCys Thr Ser Cys Arg Lys Ala 1 5 10 15 Lys Ala Trp Leu Gln Glu His AspIle Pro Tyr Thr Glu Arg Asn Ile 20 25 30 Phe Ser Glu His Leu Thr Ile AspGlu Ile Lys Gln Ile Leu Lys Met 35 40 45 Thr Glu Asp Gly Thr Asp Glu IleIle Ser Thr Arg Ser Lys Thr Tyr 50 55 60 Gln Lys Leu Asn Val Asp Ile AspSer Leu Pro Leu Gln Asp Leu Tyr 65 70 75 80 Ser Ile Ile Gln Asp Asn ProGly Leu Leu Arg Arg Pro Ile Ile Leu 85 90 95 Asp Asn Lys Arg Leu Gln ValGly Tyr Asn Glu Asp Glu Ile Arg Arg 100 105 110 Phe Leu Pro Arg Lys ValArg Thr Phe Gln Leu Gln Glu Ala Gln Arg 115 120 125 Met Val Asp 130 14321 DNA Artificial Sequence Oligonucleotide Primer 143 atttacttcaccaagttgca c 21 144 21 DNA Artificial Sequence Oligonucleotide Primer144 taattggacg acgtaataag c 21 145 20 DNA Artificial SequenceOligonucleotide Primer 145 gggcccaagc ttagtgatgg 20

What is claimed is:
 1. A method for identifying an agent that binds apolypeptide, the method comprising: contacting a polypeptide and anagent to form a mixture, wherein the polypeptide is encoded by a codingsequence comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117,121, 125, 129, 133, 137, and 141; determining whether the agent bindsthe polypeptide.
 2. The method of claim 1 wherein determining comprisesan assay selected from the group consisting of an enzyme assay, abinding assay, and a ligand binding assay.
 3. The method of claim 1further comprising determining whether the agent decreases the growthrate of a microbe, comprising: contacting a microbe with the agent;incubating the microbe and the agent under conditions suitable forgrowth of the microbe that is not contacted with the agent; anddetermining the growth rate of the microbe contacted with the agent,wherein a decrease in growth rate compared to the microbe that is notcontacted with the agent indicates the agent decreases the growth rateof the microbe.
 4. The method of claim 3 wherein the microbe is in vitroor in vivo.
 5. The method of claim 3 wherein the microbe is aStaphylococcus aureus.
 6. An agent identified by the method of claim 1.7. A method for identifying an agent that binds a polypeptide, themethod comprising: contacting a polypeptide and an agent to form amixture, wherein the polypeptide is encoded by an essential codingsequence comprising a nucleotide sequence having at least about 57percent structural similarity with a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 33, 109,113, 117, 121, 125, 129, 133, and 137; determining whether the agentbinds the polypeptide.
 8. The method of claim 7 wherein determiningcomprises an assay selected from the group consisting of an enzymeassay, a binding assay, and a ligand binding assay.
 9. The method ofclaim 7 further comprising determining whether the agent decreases thegrowth rate of a microbe, comprising: contacting a microbe with theagent; incubating the microbe and the agent under conditions suitablefor growth of the microbe that is not contacted with the agent; anddetermining the growth rate of the microbe contacted with the agent,wherein a decrease in growth rate compared to the microbe that is notcontacted with the agent indicates the agent decreases the growth rateof the microbe.
 10. The method of claim 9 wherein the microbe is invitro or in vivo.
 11. The method of claim 9 wherein the microbe is an S.aureus.
 12. An agent identified by the method of claim
 9. 13. A methodfor identifying an agent that binds a polypeptide, the methodcomprising: contacting a polypeptide and an agent to form a mixture,wherein the polypeptide is encoded by a critical coding sequencecomprising a nucleotide sequence having at least about 57 percentstructural similarity with a nucleotide sequence comprising SEQ ID NO:141; determining whether the agent binds the polypeptide.
 14. The methodof claim 13 wherein determining comprises an assay selected from thegroup consisting of an enzyme assay, a binding assay, and a ligandbinding assay.
 15. The method of claim 13 further comprising determiningwhether the agent decreases the growth rate of a microbe, comprising:contacting a microbe with the agent; incubating the microbe and theagent under conditions suitable for growth of the microbe that is notcontacted with the agent; and determining the growth rate of the microbecontacted with the agent, wherein a decrease in growth rate compared tothe microbe that is not contacted with the agent indicates the agentdecreases the growth rate of the microbe.
 16. The method of claim 13wherein the microbe is in vitro or in vivo.
 17. The method of claim 13wherein the microbe is an S. aureus.
 18. An agent identified by themethod of claim
 13. 19. A method for identifying an agent that decreasesthe growth rate of a microbe, the method comprising: contacting amicrobe with an agent, wherein the agent binds to a polypeptide encodedby a coding sequence comprising a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113,117, 121, 125, 129, 133, 137, and 141; incubating the microbe and theagent under conditions suitable for growth of the microbe that is notcontacted with the agent; and determining the growth rate of the microbecontacted with the agent, wherein a decrease in growth rate compared tothe microbe that is not contacted with the agent indicates the agentdecreases the growth rate of the microbe.
 20. The method of claim 19wherein the microbe is in vitro or in vivo.
 21. The method of claim 19wherein the microbe is an S. aureus.
 22. An agent identified by themethod of claim
 19. 23. A method for identifying an agent that decreasesthe growth rate of a microbe, the method comprising: contacting amicrobe with an agent, wherein the agent binds to a polypeptide encodedby an essential coding sequence comprising a nucleotide sequence havingat least about 57 percent structural similarity with a nucleotidesequence selected from the group consisting of SEQ ID NO: 7, 21, 23, 25,27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, and 137; incubatingthe microbe and the agent under conditions suitable for growth of themicrobe that is not contacted with the agent; and determining the growthrate of the microbe contacted with the agent, wherein a decrease ingrowth rate compared to the microbe that is not contacted with the agentindicates the agent decreases the growth rate of the microbe.
 24. Themethod of claim 23 wherein the microbe is in vitro or in vivo.
 25. Themethod of claim 23 wherein the microbe is an S. aureus.
 26. An agentidentified by the method of claim
 23. 27. A method for identifying anagent that decreases the growth rate of a microbe, the methodcomprising: contacting a microbe with an agent, wherein the agent bindsto a polypeptide encoded by a critical coding sequence comprising anucleotide sequence having at least about 57 percent structuralsimilarity with a nucleotide sequence comprising SEQ ID NO: 141;incubating the microbe and the agent under conditions suitable forgrowth of the microbe that is not contacted with the agent; anddetermining the growth rate of the microbe contacted with the agent,wherein a decrease in growth rate compared to the microbe that is notcontacted with the agent indicates the agent decreases the growth rateof the microbe.
 28. The method of claim 27 wherein the microbe is invitro or in vivo.
 29. The method of claim 27 wherein the microbe is anS. aureus.
 30. An agent identified by the method of claim
 27. 31. Amethod for decreasing the growth rate of a microbe, the methodcomprising: contacting a microbe with an agent that binds to apolypeptide encoded by a coding sequence comprising a nucleotidesequence selected from the group consisting of SEQ ID NO: 7, 21, 23, 25,27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, 137, and
 141. 32. Themethod of claim 31 wherein the microbe is in vitro or in vivo.
 33. Themethod of claim 31 wherein the microbe is an S. aureus.
 34. A method fordecreasing the growth rate of a microbe, the method comprising:contacting a microbe with an agent that binds to a polypeptide encodedby an essential coding sequence comprising a nucleotide sequence havingat least about 57 percent structural similarity with a nucleotidesequence selected from the group consisting of SEQ ID NO: 7, 21, 23, 25,27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, and
 137. 35. Themethod of claim 34 wherein the microbe is in vitro or in vivo.
 36. Themethod of claim 34 wherein the microbe is an S. aureus.
 37. A method fordecreasing the growth rate of a microbe, the method comprising:contacting a microbe with an agent that binds to a polypeptide encodedby a critical coding sequence comprising a nucleotide sequence having atleast about 57 percent structural similarity with a nucleotide sequencecomprising SEQ ID NO:
 141. 38. The method of claim 37 wherein themicrobe is in vitro or in vivo.
 39. The method of claim 38 wherein themicrobe is an S. aureus.
 40. A method for making an S. aureus withreduced virulence, the method comprising: altering a coding sequence inan S. aureus to comprise a mutation, the non-mutagenized coding sequencecomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129,133, 137, and 141; and determining if the S. arueus comprising themutation has reduced virulence compared to an S. arueus that does notcomprise the mutation.
 41. An S. aureus of claim
 40. 42. A vaccinecomposition comprising the S. aureus organism of claim
 40. 43. A methodfor making an S. aureus with reduced virulence, the method comprising:altering an essential coding sequence in an S. aureus to comprise amutation, the non-mutagenized coding sequence comprising a nucleotidesequence having at least about 57 percent structural similarity to anucleotide sequence selected from the group consisting of SEQ ID NO: 7,21, 23, 25, 27, 29, 31, 33, 109, 113, 117, 121, 125, 129, 133, and 137;and determining if the S. arueus comprising the mutation has reducedvirulence compared to an S. arueus that does not comprise the mutation44. An S. aureus of claim
 43. 45. A vaccine composition comprising theS. aureus organism of claim
 43. 46. A method for making an S. aureuswith reduced virulence, the method comprising: altering a criticalcoding sequence in an S. aureus to comprise a mutation, thenon-mutagenized coding sequence comprising a nucleotide sequence havingat least about 57 percent structural similarity to a nucleotide sequencecomprising SEQ ID NO: 141; and determining if the S. arueus comprisingthe mutation has reduced virulence compared to an S. arueus that doesnot comprise the mutation
 47. An S. aureus of claim
 46. 48. A vaccinecomposition comprising the S. aureus organism of claim
 46. 49. Anisolated polynucleotide comprising a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 33, 109,113, 117, 121, 125, 129, 133, 137, and
 141. 50. An isolatedpolynucleotide consisting essentially of a nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 33,109, 113, 117, 121, 125, 129, 133, 137, and 141, and wherein thepolynucleotide optionally further comprises from zero to up to about5,000 nucleotides upstream and/or downstream of the nucleotide sequenceselected from the group consisting of SEQ ID NO: 7, 21, 23, 25, 27, 29,31, 33, 109, 113, 117, 121, 125, 129, 133, 137, and
 141. 51. An isolatedpolynucleotide comprising a nucleotide sequence having at least about 57percent structural similarity with a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 109, 113,117, 121, 125, 129, 133, and 137, wherein the isolated polynucleotidecomprises an essential coding sequence.
 52. An isolated polynucleotideconsisting essentially of a nucleotide sequence having at least about 57percent structural similarity with a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 7, 21, 23, 25, 27, 29, 31, 109, 113,117, 121, 125, 129, 133, and 137, wherein the isolated polynucleotidecomprises an essential coding sequence.
 53. An isolated polynucleotidecomprising a nucleotide sequence having at least about 57 percentstructural similarity with a nucleotide sequence comprising SEQ ID NO:141, wherein the isolated polynucleotide comprises a critical codingsequence.
 54. An isolated polynucleotide consisting essentially of anucleotide sequence having at least about 57 percent structuralsimilarity with a nucleotide sequence comprising SEQ ID NO: 141, whereinthe isolated polynucleotide comprises a critical coding sequence.
 55. Anisolated polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 110, 114, 118, 122, 126, 130, 134, 138, and 142.